Structure and assembly of filamentous bacteriophages.

Progress in Biophysics and Molecular Biology xxx (2014) 1e43

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Progress in Biophysics and Molecular Biology journal homepage: www.elsevier.com/locate/pbiomolbio

Review

Structure and assembly of filamentous bacteriophages D.A. Marvin a, *, M.F. Symmons a, S.K. Straus b, * a b

Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Available online xxx

Filamentous bacteriophages are interesting paradigms in structural molecular biology, in part because of the unusual mechanism of filamentous phage assembly. During assembly, several thousand copies of an intracellular DNA-binding protein bind to each copy of the replicating phage DNA, and are then displaced by membrane-spanning phage coat proteins as the nascent phage is extruded through the bacterial plasma membrane. This complicated process takes place without killing the host bacterium. The bacteriophage is a semi-flexible worm-like nucleoprotein filament. The virion comprises a tube of several thousand identical major coat protein subunits around a core of single-stranded circular DNA. Each protein subunit is a polymer of about 50 amino-acid residues, largely arranged in an a-helix. The subunits assemble into a helical sheath, with each subunit oriented at a small angle to the virion axis and interdigitated with neighbouring subunits. A few copies of “minor” phage proteins necessary for infection and/or extrusion of the virion are located at each end of the completed virion. Here we review both the structure of the virion and aspects of its function, such as the way the virion enters the host, multiplies, and exits to prey on further hosts. In particular we focus on our understanding of the way the components of the virion come together during assembly at the membrane. We try to follow a basic rule of empirical science, that one should chose the simplest theoretical explanation for experiments, but be prepared to modify or even abandon this explanation as new experiments add more detail. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Fibre diffraction Inovirus Membrane transport Phage display Phyllotaxis Solid-state NMR

Contents 1.

2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Early days . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Structure determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Interesting biological features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular structure of filamentous bacteriophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Class I phage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Class II phage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. The Pf1L model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. The Pf1H model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Evidence for grouping of subunits in Pf1H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.4. Other Class II phage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.5. The Pf1 structural transition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: CS, chemical shift; MAS, magic angle spinning; PISEMA, polarization inversion spin exchange at the magic angle; PISA, polarity index slant angle; nt/su, DNA nucleotides per protein subunit; ORF, open reading frame; TB, TolA-C binding domain of p3 protein; PB, pilus-binding domain of p3 protein; LC, low-complexity domain of p3 protein; ICS, infection-competence segment of p3 protein; Tol, TolA-C; K69H, residue K at position 69 changed to H, etc.; FTIR, Fourier-transform infra-red; UVRR, ultraviolet resonance-Raman; CD, circular dichroism; FRET, Förster (or fluorescence) resonance energy transfer; IHRSR, iterative helical real space reconstruction; RF, replicative form; rmsd, root-mean-square deviation; OB-fold, oligomer-binding-fold; RPA70, human replication protein A. * Corresponding authors. E-mail addresses: [email protected] (D.A. Marvin), [email protected] (S.K. Straus). http://dx.doi.org/10.1016/j.pbiomolbio.2014.02.003 0079-6107/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Marvin, D.A., et al., Structure and assembly of filamentous bacteriophages, Progress in Biophysics and Molecular Biology (2014), http://dx.doi.org/10.1016/j.pbiomolbio.2014.02.003

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D.A. Marvin et al. / Progress in Biophysics and Molecular Biology xxx (2014) 1e43

2.3.

3.

4.

Structure refinement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. Use of Xplor-NIH for combined refinement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. Additional structural constraints from other methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Geometrical properties of subunit packing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1. Helicoid representation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2. Phyllotaxis representation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Assembly of filamentous bacteriophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Assembly at the membrane: proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. In vivo studies of assembly: proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. In vitro studies of assembly: proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.3. Models for protein assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Assembly at the membrane: DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. The p5-DNA replication/assembly complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. DNA in the virion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Supplementary material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction 1.1. Early days The filamentous bacteriophage has only a few genes and is one of the simplest biological systems known. In the half-century since it was first identified, there has been an explosion in understanding the molecular basis of the phage life cycle; and a parallel explosion in applications of the phage system to biotechnology and nanotechnology. In this extended Introduction we outline the development of the field, including a few examples of the false starts and accidental discoveries that are not usually discussed in a formal review and that illustrate Max Delbrück’s “principle of controlled sloppiness”, sometimes phrased as the motto “fail early to succeed sooner”. We also illustrate in passing how the experimental and computational techniques of fibre diffraction and solid-state NMR have improved over the last 50 years. In the late 1950s and early 1960s, interest developed in “small” phages, so-called because their genome is an order of magnitude smaller than the classical tadpole-shaped T2 and T4 phages that were then widely studied: it was thought that the small phages might be simpler and easier to understand (Sinsheimer, 1966). A few of these small phages were characterized as isometric (roughly spherical) in shape, with a diameter of about 250 A. They attracted interest not only because of their small size, but also because some of them had a single-stranded DNA genome (for instance 4X174); and some of them had a single-stranded RNA genome (for instance MS2 and f2) and were specific for male (Fþ or Hfr) strains of bacteria, which can transfer DNA to other bacteria. Several labs at this time isolated small male-specific phages without characterizing them other than to show that they had only a few genes. The first phage characterized as filamentous, rather than isometric, was fd. Hartmut Hoffmann-Berling at the Max-Planck Institute in Heidelberg became interested in small male-specific phages, and on a family outing to a favourite country restaurant, the Bierhelder Hof, he collected from the dung heap on the adjacent cattle farm a small sample which he took back to his lab. He selected for further study two plaque-formers on Hfr Escherichia coli: one, containing DNA, he named fd, and the other, containing RNA, he named fr. The fr phage was structurally similar to previously characterized isometric RNA phages. But fd phage preparations showed mixtures of elongated filamentous particles and isometric particles in electron micrographs, and this mixture was initially interpreted as contaminating bacterial pili among putative

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isometric phage. However, infectivity of these preparations was far more sensitive to ultrasonication than control preparations of similar-sized isometric phage, as expected if the elongated particles are in fact the phage (Fig. 1). Physicalechemical characterization confirmed the general features of filamentous fd phage and its DNA (Marvin and Hoffmann-Berling, 1963a,b). Other researchers looked at their own small phages, which they had thought to be isometric, and some of these turned out to be filamentous phages as well, notably f1, which was isolated at the same time as the isometric RNA phage f2, but not further characterized at the time (Loeb and

Fig. 1. Inactivation of fd filamentous phage by ultrasonication, relative to isometric phage of similar size. Mixtures of fd with control phage (either fr or 4X174), 1011 to 1012 plaque-forming units/ml in each case, were sonicated for the indicated time and plated on an Fþ strain of E. coli C (which is sensitive to all three phages). The different phages could be distinguished by their different plaque types. Circles, fd mixed with 4X174; rectangles, fd mixed with fr. Filled symbols, fd; open symbols, control phage. First reported in Nature on 2 February 1963 (Marvin and Hoffmann-Berling, 1963a).



Zinder, 1961; Zinder et al., 1963; Zinder, 1986). One researcher, having found a phage that looked similar to fd (Hofschneider, 1963) went on tour giving talks about his phage, M13, and distributing samples of the phage. In consequence many labs started to work on M13; whereas those with roots in f1 phage continued with f1. The three phages fd, f1 and M13 have about 98.5% DNA sequence identity and for most purposes they can be considered to be identical. These three phages and filamentous phage ZJ/2 (Bradley, 1964), which has been less widely studied, are all specific for E. coli bacteria expressing F-pili (the tip of the F-pilus is the attachment site for the phage; see, for example, Bayer and Bayer, 1986). Therefore the name Ff, for F-specific filamentous phages, is sometimes applied to members of this group of phages, to distinguish them from other filamentous phages with similar architecture, but different detailed genetics and physiology. Table 1 lists some coat protein sequences as markers for the relationship between filamentous phage strains. Within the Ff group, phage fd has been used more for structural studies, and phages f1 and M13 for physiological and genetic studies. The fd genome was the first filamentous phage genome to be sequenced (Beck et al., 1978), and in fact only the second DNA genome ever sequenced, after the isometric phage 4X174 (Sanger, 1975). The fd sequence showed that the 6408 nucleotides can be assigned to a small number of open reading frames, plus an intergenic non-coding sequence into which foreign genes can be inserted to create a cloning vector (Herrmann et al., 1978, 1980; Schaller, 1979). In preliminary X-ray fibre diffraction studies of fd structure, samples were prepared by aligning concentrated gels of purified phage by flow in capillaries, a method that is successful with large rigid rod-shaped viruses such as tobacco mosaic virus. But fd has only about one tenth the cross-sectional area of tobacco mosaic virus, and is more flexible and less amenable to aligning by flow.

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The fd phage in capillaries were in fact arranged with their long axes perpendicular, not parallel, to the long axis of the capillary. This became apparent from studies of the fibre diffraction pattern as a function of water content, which showed that lattice spacings expanded equally in all directions with increasing water content, whereas for phage aligned along the capillary axis one would expect the spacings between phage to expand much more than the spacings along the phage length. True alignment was achieved with a technique used for fibre diffraction of nucleic acids: a drop of a concentrated but unaligned gel of fd is hung between the tips of two glass rods, and allowed to dry into a fibre, with slight stretching (Marvin, 1966). The overall distribution of intensity on X-ray diffraction patterns of fd fibres (Fig. 2) is typical of an array of a-helices with the long axes of the helices oriented roughly parallel to the long axis of the fibre. Strong intensity at about 10 A spacing in the equatorial direction (roughly perpendicular to the fibre axis) is attributed to the distance between close-packed a-helices, which are about 10 A in diameter; and strong intensity at about 5.4 A in the meridional direction (roughly parallel to the fibre axis) is attributed to the 5.4 A pitch of the a-helix. These features suggested that the coat protein subunit of the virion is largely a-helical, with the a-helix axis roughly parallel to the virion axis (Marvin, 1966). Loren Day confirmed that optical rotatory dispersion measurements are also consistent with a largely a-helical fd coat protein (Day, 1966). Surprisingly for a linear virus, the DNA isolated from the fd virion has a single-stranded circular topology. This was demonstrated by measuring both the sedimentation and viscosity of fd DNA solutions in a solvent that promotes extended chains, as a function of biological inactivation by pancreatic DNase. A single “hit” of biological inactivation was equated to a single break in the DNA chain. The viscosity increased to a maximum at about

Table 1 Amino acid sequences of Inovirus p8 major coat proteins.


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Fig. 2. X-ray fibre diffraction patterns of fd and Pf1. The fibre axis direction (meridional direction) is vertical, and the equatorial direction is horizontal. The strong diffracted regions in the w10 A equatorial direction and the w5.4 A meridional direction, typical of an a-helix, are indicated, as is the near-meridional layer line intensity indicating the pitch repeat of w15 A for Pf1 and w16 A for fd. Note that in diffraction studies there is a reciprocal relationship between spacings in the sample and spacings on the diffraction pattern. Samples were magnetically aligned. The diffraction patterns have been mapped from detector space to reciprocal space (hence the blank non-observed portions of the meridional region), quadrant averaged, and mirrored across the equator. The outer edges of the diffraction patterns coincide with the silicon powder calibration ring at 3.136 A. The two patterns are shown to the same reciprocal space scale. We are grateful to Dr Liam Welsh for supplying the pair of processed diffraction patterns. See Supplementary material, Section S1, for more detailed interpretation of X-ray fibre diffraction patterns.

one hit and the sedimentation coefficient decreased slightly. If a solution of linear polymer molecules receives one random hit per molecule, the weight average molecular weight will be roughly halved, and the sedimentation coefficient and the viscosity will both decrease. But if a circular molecule receives one hit, its molecular weight will be unchanged, while the molecule can become more extended. In this case, the viscosity will be greater than that of the intact molecule, although the sedimentation coefficient may be less (Marvin and Schaller, 1966). This increase in viscosity has not been demonstrated for the DNA of other filamentous phage, notably for Pf1, but circularity of filamentous phage DNA has often been assumed by analogy with fd (see also Section 3.2.2). The circular topology suggests that a single strand of DNA runs from one end of the virion to the other and then back again, like a circle of string pulled taut from opposite sides of its circumference. This fact, combined with electron micrographs indicating two parallel strands in the intact phage (Marvin, 1966), suggested a model for the phage structure with each strand of the DNA separately encapsidated in a bundle of a-helices, and these two nucleo-protein rods lying parallel to one another to form the virion. But following detailed analysis of the equatorial diffraction pattern, showing that the cross-section of the phage is roughly circular with diameter about 60 A, comprising a protein sheath surrounding a DNA core (Wachtel et al., 1974), the two-strand model was abandoned in favour of the more conventional single rod model proposed by Bradley (1964).

The arrangement of protein subunits in fd phage still remained elusive. To gain more information, X-ray fibre diffraction patterns of other strains of filamentous phage were examined. This survey showed that strains of filamentous phage can be separated into two structural classes on the basis of their diffraction patterns. The two classes have a similar overall distribution of diffracted intensity, indicating a similar structure for the subunit; but they have somewhat different layer line positions, indicating different detailed symmetries relating the subunits (Marvin et al., 1974b), so the diffraction pattern is an immediately recognizable “fingerprint” of the structural class (Fig. 2). Class I is exemplified by the fd strain and the other Ff phage strains, but is not limited to the Ff strains and includes other phages such as IKe and If1, which have different coat protein sequences and do not adsorb to F-pili (Marvin et al., 1974a). Class II is exemplified by the Pf1 strain, and includes phages Xf, Pf3 and PH75. Thomas and Murphy (1975) used spectroscopic studies to confirm for Pf1, as Day had done for fd, that the coat protein is largely a-helical. Filamentous bacteriophage of both classes (Table 1) are members of the genus Inovirus (Ackermann, 2006). Class I phage grow in E. coli bacteria; Class II phage grow in other Gram-negative species. The structural class of phage B5 (Chopin et al., 2002), that grows in Gram-positive bacteria, has not yet been determined. In the early studies, it was assumed that filamentous phage had only one type of coat protein, like many other simple viruses. This protein was initially called the “B protein” but later assigned to gene 8 and therefore called p8. Analysis of mutants by Pratt et al. (1966, 1969) showed that the virion contains at least one other type of coat protein, initially called the “A-protein” but now called p3, present in only a few copies in the virion, that is important for morphogenesis and infection (see Section 3.1.1.2 for further discussion). Some other questions of nomenclature should be noted. Pratt et al. (1966) in their initial genetic studies used Arabic numerals (1, 2, 3, 4 .) to represent the M13 phage genes. Lyons and Zinder (1972) in their studies of f1 mutants used the same relationship between gene and number as Pratt et al. (1966), but used Roman numerals (I, II, III, IV.) instead of Arabic numerals. This difference is still maintained by some researchers, depending on their scientific background. Here we use Arabic numerals throughout to represent genes for all filamentous phage, and translate from Roman to Arabic where necessary. We refer to the mature protein product of fd gene 8 (after removal of the leader peptide) as p8. Other notations found in the literature (pVlll, gVlllp, GVIIIP, g8p, gp8, etc.) are converted here to this notation and we use a corresponding notation for other gene products (p3, p5, p6, etc.) and for analogous proteins from other filamentous phage strains. 1.2. Structure determination The Pf1 strain proved easier than fd to characterize structurally because a slight symmetry difference means that diffraction data that are superposed in fd diffraction patterns are resolved for Pf1. Direct analysis of the qualitative intensity distribution on diffraction patterns of Pf1 showed that, unlike other previously studied viruses, the coat protein subunits are elongated, not globular, with their long axes at a small angle to the virion axis (see Section S.1 of the Supplementary material for details of the analysis), and the virion coat is an overlapping interdigitated array of these subunits (Marvin et al., 1974b). To enable quantitative analysis of the Pf1 diffraction data, Ellen Wachtel and later Jane Ladner and Colin Nave wrote computer programs that process data from digitized continuous transform diffraction patterns, correcting for continuous background and other artifacts (a quite different problem than for single-crystal data) to produce continuous transform observed



data that for the first time could be directly compared with the continuous Fourier transforms calculated from detailed molecular models (Wachtel et al., 1974; Nakashima et al., 1975; Marvin and Wachtel, 1975, 1976; discussed in more detail by Marvin et al., 1987). The molecular model for Pf1 was refined and extended to other strains as more and better data became available, and has now been thoroughly confirmed as the architecture of filamentous bacteriophage. The amino-acid sequence (Table 1) of the Pf1 major coat protein has several negatively charged amino acids in the N-terminal region, is rich in hydrophobic amino acids in the central region, and has several positively charged residues in the C-terminal region, like the corresponding regions of the fd coat protein sequence. The centre-to-centre distance between virions in dry fibres (a measure of the diameter of the virion) is about 55 A for both fd (Marvin, 1966; Wachtel et al., 1974) and Pf1 (Marvin et al., 1974b). The overall intensity distributions on the Pf1 and fd diffraction patterns are similar, as is the near-meridional spacing (15 A for Pf1 and 16 A for fd). These similarities (Fig. 2) suggest that the fd and Pf1 virion structures are similar, with the arrangement of subunits in fd essentially a subtly modified version of that in Pf1 (Marvin et al., 1974a). The protein sheath comprises thousands of elongated slightly curved a-helical major coat protein subunits, each about 70 A long by 10 A diameter, with the subunit axis oriented at a small angle to the virion axis and also sloping radially, to form an overlapping interdigitated helical array. A schematic representation of the Ff virion is illustrated in Fig. 3. Molecular model-building and Fourier transform calculations supported the general structural model for the Pf1 virion, but it was several more years before the symmetry relationship between fd and Pf1 was fully resolved. The actual structures of the two symmetry classes are very similar, and in fact are difficult to distinguish at low resolution (Fig. 4). A description of the symmetry that emphasizes this similarity is given by Marvin (1990). Although the specific fd symmetry initially proposed by Marvin et al. (1974a) was later modified as better data became available (Caspar and Makowski, 1981; Banner et al., 1981), the notion of a close relationship between fd and Pf1 symmetry led to discussion that a structure of this type could have two inter-convertible states, and this might be of more general importance in structural molecular biology (Marvin et al., 1974a; see also Section 2.2.5). During a search for conditions to improve X-ray patterns of Pf1, that included drying fibres in a fridge, Fizz Marvin found a slightly different X-ray fibre

Fig. 3. Schematic representation of Ff phage. The protein sheath comprises thousands of elongated slightly curved a-helical major coat protein subunits, each about 70 A long by 10 A diameter, with the subunit axis oriented at a small angle to the virion axis and also sloping radially, to form an overlapping interdigitated helical array, variously compared with shingles or roof tiles or thatching, or fish or snake-skin scales. The proximal p3:p6 adsorption complex is at the left and the distal p7:p9 complex with the double-stranded DNA packaging signal is at the right. Apart from the packaging signal, the DNA is single-stranded but, as shown, the circular single-stranded molecule runs from one end of the virion to the other and back again to complete the circle. Except for the packaging signal, the two strands are not base-paired to each other. The Cterminus of the coat p8 (shown shaded blue) is rich in basic residues that interact with the DNA.

5

Fig. 4. Subunit arrangement in the two classes of filamentous bacteriophage. The virion axis is vertical, showing an axial slab corresponding to about 1e2% of the total length of the virion. Each subunit is represented as a space-filling coil following the protein backbone at 5 A radius. Left: fd (class I). Right: Pf1 (class II). Formally, the symmetry of fdD is C5S2 and the symmetry of Pf1H is C1S5.4. This figure illustrates how the formal symmetry description, which suggests quite different structures, can be misleading. After Marvin (1990); see also Welsh et al. (2000) and Marvin et al. (2006).

pattern, and Ellen Wachtel realized that this was an example of such inter-convertible states (Wachtel et al., 1976). Unfortunately the fridge used during the continued search for improved X-ray patterns failed to reach the low temperatures of the original experiment, and this failure led efforts to be diverted to examining other conditions such as ionic strength and pH. Eventually Herbert Siegrist repeated the low temperature experiment properly, and found that above about 283 K the phage is in the “higher temperature” form, Pf1H, but below this temperature, the phage is in the “lower temperature” form, Pf1L (Nave et al., 1979). To confirm that this temperature-induced transition was truly an intrinsic property of the virion, and not just some fibre or liquid crystal artifact, Hinz et al. (1980) studied the transition using various solution techniques. This transition is interesting not only in itself, but also because the better resolution (layer-line splitting) of the Pf1L diffraction patterns gives true three-dimensional data to about 3 A resolution along the layer-lines. In 1975, Stan Opella at MIT did some NMR experiments on a sample of fd suggesting that this would be a good research object for the solid-state NMR lab he was about to set up at the University of Pennsylvania, and in 1981 his colleague Tim Cross visited EMBL Heidelberg to learn about aligning purified samples of filamentous phage (Cross and Opella, 1983; Cross et al., 1983). The NMR studies led to a pair of cover stories in the 31 May 1991 issue of Science: Shon et al. (1991) interpreted solid-state NMR experiments on isolated Pf1 coat protein reconstituted into lipid bilayers to suggest that the precursor coat protein consists of two a-helical segments, one spanning the membrane and the other lying parallel to the outer surface of the membrane; and Nambudripad et al. (1991) interpreted 8 A resolution neutron diffraction data from fibres of Pf1 deuterated in specific amino acid sidechains to suggest that the protein subunit in the virion consists of two a-helix segments, with residues 13 to 19 in a disordered non-helical surface loop. These models took the field on a false scent. Vos et al. (2009) and Hemminga et al. (2010) reviewed the extensive evidence that


6


discounts the two-segment model for the coat protein in the membrane. The evidence discounting the surface loop for the coat protein in the virion is also extensive. Electron density maps calculated using 3.3 A resolution native and single isomorphous derivative X-ray diffraction data from Pf1 are not consistent with a surface loop (Gonzalez et al., 1994, 1995). Solid-state NMR measurements of specifically labelled aligned phage (Opella et al., 2008) confirm the absence of any significant disordered non-helical loop or break within the subunit a-helix of either Pf1 or fd, except for a few residues at the N-terminus. Refining the structure against the NMR and the X-ray fibre diffraction information in parallel gave consensus models for fd (Marvin et al., 2006) and for Pf1 (Straus et al., 2011). For X-ray fibre diffraction and solid-state NMR studies, it is desirable to maximize the alignment of molecules within the fibre, and filamentous phage can be aligned by shear to a disorientation half-width of about 10e15 . Much effort was expended to search for ways to improve the alignment. The observation by Samulski and Tobolsky (1971) that a-helices can be aligned by a strong magnetic field led various colleagues at various times in the 1970’s, but unsuccessfully and never published, to try to align filamentous phage in a magnetic field. Finally Jim Torbet, working with Georg Maret at the High-Field Magnet Laboratory of the Max Planck Institute in Grenoble, undertook a systematic study of magnetic alignment of the phage, using facilities that allow quantitative measurement of alignment as a function of field strength, phage concentration and other parameters, and found that indeed it was possible to align Pf1 and fd using a magnetic field (Maret et al., 1979; Torbet and Maret, 1981; Maret and Dransfeld, 1985; Maret, 1990). Torbet and Maret (1979) also made fibres of Pf1 in a strong magnetic field, although their diffraction patterns were not as good quality as the best patterns from fibres previously obtained without magnetic alignment. Alan Fowler further developed magnetic alignment of purified liquid crystalline phage, to give Pf1L fibre diffraction data with a disorientation half-width of 1.5 , comparable to 3.3 A resolution single-crystal data (Nave et al., 1979). Magnetically aligned samples of filamentous phage have also proven to be useful as matrices to align other molecules in solutionstate NMR experiments, using either Pf1 (Hansen et al., 1998) or fd (Clore et al., 1998). Attempts to generate heavy atom derivatives of fd fibres by soaking fibres in solutions of heavy atoms proved difficult, because the fibres lost alignment. Using osmium tetroxide vapour on aligned fibres (Wachtel et al., 1974) caused intensity changes on the diffraction patterns, but did not indicate specific isomorphous substitution of heavy atoms. Nakashima and Konigsberg (1980) showed that it was possible to iodinate one of the Tyr subunits on the Pf1 coat in solution, from which fibres could be subsequently prepared, and calculations suggested that fibres of these iodinated samples might show measurable intensity changes on the diffraction patterns. Using magnetically aligned samples of Pf1L it was possible for the first time to detect the small intensity changes on X-ray patterns introduced by heavy atom derivatives of Pf1. Pf1 derivatives prepared by Ray Brown, Akira Tsugita, John Armstrong and Richard Perham were aligned in fibres, measured and analysed by Alan Fowler, Colin Nave and Jane Ladner to resolve some questions about details of Pf1 symmetry and to confirm the general properties of the Pf1 model (Nave et al., 1981). Both Tyr residues in wild-type Ff phage coat protein are accessible to iodination, so in order to enable unique iodinated derivatives in this phage strain, Manuela Helmer-Citterich prepared Y21M and Y24M mutants (Marvin et al., 1994), and iodinated derivatives of such mutants were used to determine the radii of Y24 and Y21 (Marvin et al., 2006). Serendipitously, Y21M also shows a better quality diffraction pattern than wild-type (Marvin et al., 1994), which may in part

be due to the fact that the Y21M virion appears more rigid than wild-type fd (Barry et al., 2009) and therefore easier to align. Model-building and intensity measurement confirmed that the axes of the a-helical subunits are oriented at a small angle (about 20 ) to the axis of the phage, which implies that neighbouring subunits cross each other at a small angle; and the sign of this angle defines the orientation with respect to the phage axis (Marvin and Wachtel, 1975, 1976). This crossing angle was initially chosen to have the same sign as found in coiled-coil a-helices (Crick, 1953; Lucas and Lambin, 2005), implying that the a-helix axis winds around the phage axis in a left-handed sense, although it was recognized (Marvin and Wachtel, 1975) that this choice needed experimental verification. Richard Bryan developed a Maximum Entropy algorithm that uses Pf1 native and single isomorphous derivative fibre diffraction data to calculate an unbiased electron density map that fits the native and derivative data as well as possible without introducing unjustified detail. He found that this map appeared to show that the a-helix is a left-handed helix, contrary to fact. The way out of this dilemma was to propose that the original choice of enantiomorph was incorrect: that is, the ahelix axis winds around the phage axis in a right-handed rather than a left-handed sense (Bryan et al., 1983; Bryan, 1987; Marvin et al., 1987). Further refinement against high resolution X-ray fibre diffraction Pf1 data enabled quantitative refinement of models (Marvin et al., 1992; Gonzalez et al., 1994, 1995; Straus et al., 2011). Models of fd were subsequently built assuming the same subunit orientation as Pf1. Analysis of solid-state NMR data confirmed this orientation for both Pf1 and fd phage, and thereby defined the hand of the helix relating the protein subunits in both phage (Straus et al., 2008a). Determining the hand of Pf1 phage and fd phage independently is not as trivial as it may at first appear, in view of the fact that the helical intracellular replication/assembly complex between phage DNA and the intracellular replication-assembly protein p5 is left-handed for fd, but right-handed for Pf1 (see Section 3.2.1 for further discussion). 1.3. Interesting biological features The fd phage are released from infected bacterial cells without lysis of the cells, unlike classical bacteriophage such as the T-even phage, and other small phages such as 4X174. Instead, fd phage are released from infected cells as the cells continue to grow and divide. This was shown by Hoffmann-Berling et al. (1963) using the classical single-burst method (Ellis and Delbrück, 1939), in which a suspension of infected bacteria is diluted and distributed among a large number of samples, each containing on average less than one infected bacterium. After further incubation, single fd-infected bacteria released phage, but also (unlike the classical experiments) the samples contained multiplying bacteria. HoffmannBerling and Mazé (1964) confirmed this result by the micromanipulation technique of Lwoff and Gutmann (1950), which also showed that individual fd-infected, growing bacteria liberated phage while the infected bacteria continued to multiply. These experiments, combined with the evidence that the coat protein subunit in the phage is an elongated a-helix, led to the suggestion that filamentous phage is assembled as it extrudes through the bacterial membrane, with the hydrophobic a-helical coat protein initially spanning the plasma membrane and assembling around the DNA during extrusion of the phage (Marvin and Hohn, 1969; Marvin and Wachtel, 1976). The proposal that the major coat protein is a membrane-spanning protein for part of its life cycle has been extensively supported (reviewed by Wickner, 1988). How is the phage single-stranded circular DNA, which by itself in solution is a random coil with hairpin loops, presented to the



membrane in a linear form that can be encapsidated by the coat protein into the linear phage? This question was resolved by David Pratt and colleagues, using the method of conditional lethal mutants developed with phage T4 (Epstein et al., 1963). Randomly isolated Ff phage defective mutants can be grouped into genes by genetic complementation. In general, two mutants defective in different genes can, between them, supply all phage functions, and will produce phage in an infected cell; two mutants in the same gene will not produce the corresponding gene product and will not produce phage (Pratt et al., 1966, 1969; Lyons and Zinder, 1972). Bacteria infected with a conditional lethal phage strain under permissive conditions will produce the gene product; under non-

Table 2 Molecular models of Inovirus proteins. Entrya

Strainb

Protein

Methodc

2C0W 2C0Xd

fd fd

p8 p8

1IFK 1IFL 2XKMe

If1 IKe Pf1L

p8 p8 p8

1QL1f 1QL2f

Pf1H Pf1H

p8 p8

1IFPg 1HGVh 1HGZh 2IFO 2G3P 1TOL 2 9B 2X9A 4EO0 4EO1 4G7W 4G7X 1GVP 1VQB 1GPV 1GKH 1VQI 1VQJ 1VQA 1VQF 1VQH 1VQG 1VQD 1VQC 1VQE 1AE3 1AE2 1YHB 1YHA 2GVB 1PFS

Pf3 PH75 PH75 Xf fd:TB þ PB fd:TB þ Tol If1:TB If1:TB þ Tol IKe:PB IKe:TB CTX4:TB CTX4:TB þ Tol f1 f1 f1þDNA f1-K69H f1-I47V f1-V35I f1-V35A,I47L f1-V35I,I47V f1-I47M f1-I47L f1-V35I,I47L f1-V35I,I47F f1-V35I,147M f1-R82C f1-L32R f1-Y41F f1-Y41H M13-Y41H Pf3

p8 p8 p8 p8 p3 p3 p3 p3 p3 p3 p3 p3 p5 p5 p5 p5 p5 p5 p5 p5 p5 p5 p5 p5 p5 p5 p5 p5 p5 p5 p5

X-ray fibre diffraction, 3.2 A X-ray fibre diffraction, 3.2 A, and solid-state NMR Modelling þ X-ray fibre diffraction, 5 A Modelling þ X-ray fibre diffraction, 5 A X-ray fibre diffraction, 3.3 A, and solid-state NMR X-ray fibre diffraction, 3.1 A X-ray fibre diffraction (perturbed model), 3.1 A X-ray fibre diffraction, 3.1 A X-ray fibre diffraction, 2.4 A X-ray fibre diffraction, 2.4 A Modelling X-ray diffraction, 1.9 A X-ray diffraction, 1.85 A X-ray diffraction, 2.9 A X-ray diffraction, 2.5 A X-ray diffraction, 1.6 A X-ray diffraction, 1.8 A X-ray diffraction, 2.9 A X-ray diffraction, 1.4 A X-ray diffraction, 1.6 A X-ray diffraction, 1.8 A Modelling X-ray diffraction, 2.0 A X-ray diffraction, 1.8 A X-ray diffraction, 1.8 A X-ray diffraction, 1.8 A X-ray diffraction, 1.8 A X-ray diffraction, 1.8 A X-ray diffraction, 1.82 A X-ray diffraction, 1.82 A X-ray diffraction, 1.8 A X-ray diffraction, 1.8 A X-ray diffraction, 2.0 A X-ray diffraction, 2.0 A X-ray diffraction, 2.2 A X-ray diffraction, 2.5 A Solution NMR Solution NMR

d

a

From the Protein Data Bank. PB ¼ pilus binding domain, TB ¼ TolAeC binding domain, Tol ¼ TolA-C, K69H ¼ residue K at position 69 changed to H, etc. c For X-ray fibre diffraction, more recent models of a given phage strain do not necessarily replace earlier models but are instead sometimes intended for comparison of different refinement methods. d The continuous transform X-ray fibre diffraction native data used to determine these structures are on deposit as PDB entry R2C0WSF. e The continuous transform X-ray fibre diffraction native data used to determine this structure are on deposit as PDB entry R2XKMSF. f The continuous transform X-ray fibre diffraction native data used to determine these structures are on deposit as PDB entry R1QL1SF. g The continuous transform X-ray fibre diffraction native data used to determine this structure are on deposit as PDB entry R1IFPSF. h The continuous transform X-ray fibre diffraction native data used to determine these structures are on deposit as PDB entry R1HGVSF. b

7

permissive conditions (which may be a non-permissive bacterial strain or a non-permissive growth temperature) it will not produce the gene product. Differential labelling of intracellular proteins under the two conditions can therefore identify a particular gene product and give information about its function. Surprisingly, gene 5 produces an intracellular protein in an amount comparable to the amount of the major coat protein, the product of gene 8 (Henry and Pratt, 1969), and the p5 protein is necessary for phage production (Salstrom and Pratt, 1971). Alberts et al. (1972) purified this p5 protein and showed that it binds to single-stranded DNA. The p5 protein is present in the infected cell in a complex with phage circular single-stranded DNA, in an elongated intracellular nucleoprotein complex (Pratt et al., 1974). Unlike p8, the p5 protein is soluble (as a dimer) in aqueous solvents, but attempts to determine its crystal structure proved to be more difficult than expected. McPherson et al. (1979) proposed a model based on a multiple isomorphous replacement map at 2.3 A, but it was difficult to refine this map. Brayer and McPherson (1983) showed that there were flaws in the tracing of the chain through this map, and proposed a new model that could be refined further, but this model proved to be flawed as well. Further studies by solution NMR (Folkers et al., 1994) and X-ray crystallography with multiwavelength anomalous diffraction (Skinner et al., 1994; Su et al., 1997) led to the currently accepted model of p5 structure, reviewed by Stassen et al., 1995 and Konings et al., 1995; see also Table 2. Many single or double point mutations in the wild-type p5 protein have been isolated and studied by X-ray crystallography, and these results have been used to analyse the effect of mutations on aspects of the protein such as the interface between the protein dimers, the DNA binding properties, and the hydrophobic protein core. The p5 protein structure has also been used to generate models of the linear complex of p5 and single-strand DNA, and is discussed further in Section 3.2.1. Experiments on the relative orientation of the virion with respect to the membrane during infection and during assembly suggest that the orientation of the membrane-spanning coat protein, and therefore the orientation of the virion with respect to the membrane, is the same during both infection and assembly. But in early studies, Jazwinski et al. (1973) proposed that both infection and assembly require a specialized “pilot protein” to start the DNA across the membrane. At the time the only known minor protein in the virion was p3, which was required for infection. If the same protein is used as a pilot protein for both infection and assembly, then the polarity of the virion would be reversed between infection and assembly. Lopez and Webster (1983) studied this question experimentally. They showed that different minor proteins (p7 and p9), which are at the end of the virion distal to p3, exit first from the cell during assembly, and therefore probably form the suggested “pilot protein” for assembly. The current view is that the orientation of the virion is the same during both infection and assembly (reviewed by Wickner, 1988). The p7 and p9 minor proteins are involved in the initiation of assembly, in association with a specific hairpin loop in the viral DNA. At the end of the virion that is assembled last, the p3 and p6 proteins form a knob-and-stalk structure (Gray et al., 1981) which is also required for adsorption to the pili of the host during the infective cycle. The four minor proteins are present in only a few copies each, and make up only about 2% of the total weight of the virion, so X-ray diffraction studies of the whole virion do not detect them. The role of the minor coat proteins in assembly is discussed in more detail in Section 3.1. As well as the minor proteins capping the two ends of the phage, other bacterial and phage proteins, including phage proteins p1, its restart partner p11, and p4, are necessary in the membrane for Ff assembly (Fig. 5). The role of these non-virion proteins in phage


8


useful but can also be dangerous, because the transferred DNA may encode virulence systems which can create pathogenic strains of bacteria (Davis and Waldor, 2003; Derbise et al., 2007). The review by Hemminga et al. (2010) discusses some aspects of phage assembly at the membrane. 2. Molecular structure of filamentous bacteriophages

Fig. 5. Filamentous phage Ff assembly at Gram-negative membranes. Before assembly the phage DNA is associated with the p5 protein (green) except in the region of the packaging signal. The packaging signal binds a membrane-associated complex of p7:p9 at the start of assembly. Assembly continues as shown with the membrane-bound p8 becoming associated with the DNA and being transferred across the inner membrane (IM) and outer membrane (OM). A complex of p1 and p11 (black wedge) with putative ATPase activity is involved in the assembly at the inner membrane. A multimer of p4 forms a pore in the outer membrane. Once all the DNA has dissociated from p5, assembly is completed by the addition of p3:p6 from the membrane.

assembly has been extensively studied by Marjorie Russel and others and is reviewed by Russel et al. (1997), Webster (2001) and Russel and Model (2006). Comparison of the fd and Pf1 genomes reveals that some genes of fd and Pf1 have similar functions (coding for major and minor coat proteins and for the intracellular replication-assembly protein p5), and even appear to have much the same order on the genome; but the DNA sequences within the genes show no significant relatedness, even when they code for proteins with similar properties (Nakashima et al., 1975; Hill et al., 1991). The X-ray diffraction patterns of fd and Pf1 show that the p8 coat protein subunits have similar secondary structures, and similar orientation and local packing within the virion, but both the virion symmetry and the DNA-protein interactions are different in the two virions. Both viruses form intracellular replication-assembly nucleoprotein complexes between the phage DNA and the p5 protein, and again these complexes are similar in design, but differ in detail (Gray et al., 1982a,b; Gray, 1989). The p8 coat proteins of both fd and Pf1 are initially synthesised with an N-terminal “signal sequence” of amino acid residues that are cleaved off in vivo as the coat protein is inserted into the membrane; but Class II strains Pf3 and PH75 do not have this p8 signal sequence, and thus may be more suitable for in vitro reconstitution experiments. Different strains of filamentous phage have a similar overall life cycle, but differ in detail. By comparing the details one can increase understanding of the significant features of the life cycle. The similarities and differences between p8 amino acid sequences of various filamentous phage strains are compared in Table 1. The physiology and genetics of filamentous bacteriophages have been reviewed by Webster (2001), Russel and Model (2006) and Rakonjac et al. (2011), among others. The book by Sambrook and Russell (2001) describes the use of filamentous phage for molecular cloning; see also the BioBrick strategy (Anderson et al., 2010; Norville et al., 2010). Filamentous phages can carry foreign DNA inserted into their own genome, and express the foreign DNA in bacteria (Hermann et al., 1978, 1980; Smith, 1985), which can be

As outlined in Section 1.2, the fd-type fibre diffraction pattern, defined as Class I, is observed not only for Ff phage but also for some other phage strains such as IKe and If1; whereas the similar but distinct Class II diffraction pattern is observed not only for Pf1 phage but also for phages Xf, Pf3 and PH75. The Class I diffraction pattern initially eluded interpretation, but the molecular architecture of the Class II filamentous bacteriophage protein coat was determined by direct analysis of the distribution of intensity on Pf1 X-ray fibre diffraction patterns (Marvin et al., 1974b). In particular, the distribution in the strong 10 A near-meridional region of intensity shows that the subunit is not a globular protein, but is elongated, with its long axis at a small angle to the virion long axis; the experimental evidence for this is discussed in more detail in the Supplementary material, Section S1. The phage structure was refined by building molecular models to compare with quantitative X-ray fibre diffraction and solid-state NMR data. Coordinates of molecular models of the p8 protein subunit, and the symmetry operators relating p8 subunits in the coat protein array, have been deposited in the Protein Data Bank; the corresponding entry numbers are listed in Table 2. For more recent models, the continuous transform X-ray fibre diffraction data has also been deposited, as noted in the table. Readers unfamiliar with X-ray fibre diffraction and/or solidstate NMR may want to refer to the Supplementary material for help with some of the technical details discussed in this section. 2.1. Class I phage The diffraction pattern of fd shows strong meridional reflexions at orders of about 16 A, with layer-lines at orders of about 32 A, suggesting that the p8 subunits in the phage are related by a twofold screw axis along the virus helix with a pitch of 32 A. The mass per length of fd phage and the mass of the protein subunit indicates A repeat, so the symmetry can be about 5 protein subunits per 16 described as a 5-fold rotation axis combined with an approximate two-fold screw axis (Caspar and Makowski, 1981; Banner et al., 1981). Wild-type fd, Ike and If1 in standard conditions deviate slightly from two-fold screw symmetry (1.97 instead of 2.00 units per 16 A turn, Marvin et al., 1994), and this is defined as the “canonical” fdC symmetry, typified by the molecular model deposited in the Protein Data Bank as PDB ID: 1IFD (Marvin, 1990). The exact two-fold screw symmetry found for wild-type Ff below its isoelectric point (Banner et al., 1981), and also for the D12N mutant (Bhattacharjee et al., 1992) and for the Y21M mutant (Marvin et al., 1994), is defined as the “diad” symmetry, fdD. The Class I structure was interpreted by molecular replacement from Class II, assuming a model for the structure of Class I based on a perturbed Class II helix (Marvin et al., 1974a). The Class I model was further refined as improved X-ray data and further structural information became available, leading to the structure deposited as PDB ID: 2C0W (Marvin et al., 2006). NMR methods give information about the structure of individual p8 subunits in the phage, but not about the packing relations between these subunits (see Section 2.3). Since the phage is too large to permit the rapid tumbling needed for solution-state NMR, solid-state NMR methods were used. The methods used included both oriented methods and magic angle spinning (MAS) of the



sample, described in more detail in the Supplementary material, Section S2. In both cases, isotopically labelled samples are required to obtain the necessary spectral information. These methods can give important information about a-helix structures. First, they can help to define the regions of the protein that are a-helical; and second, for oriented methods, they can define both the orientation of the a-helix axis with respect to the virion axis, and the azimuthal orientation of the a-helix around its own axis, thereby helping to define the interfaces between subunits in the protein coat. The early solid-state NMR studies on the fd coat protein (Cross and Opella, 1983, 1985) were one-dimensional and yielded 15N chemical shift and 15Ne1H dipolar splitting values. These data were used to determine the structure of the segment from residue 40e45 in the p8 sequence, Table 1 (Lys-Leu-Phe-Lys-Lys-Phe). The amide backbone of p8 was labelled at selected sites by adding 15N-labelled amino acids to the growth medium during phage preparation, namely Leu (L14, L41), Phe (F11, F42, F45) and Lys (K8, K40, K43, K44, and K48). In order to distinguish the different labels, Cross and Opella (1985) used, in addition, samples where residue i is labelled at the carbonyl position with 13C and residue i þ 1 is labelled with 15 N at the amide. By knowing where the 13C label is introduced and the primary sequence, they obtained spectra where one of the 15N lines changed from a single line to an overlap of a singlet and doublet (due to incomplete labelling) and could therefore be uniquely assigned to the Leu and Phe resonances. The Lys residues, on the other hand, were difficult to resolve and therefore not uniquely assigned. This study concluded that the segment from residue 40e45 is a-helix. These initial studies of fd p8 structure by solid-state NMR (reviewed by Opella et al., 1987) were followed by studies on the dynamics of p8 in the virion (Colnago et al., 1987). Tan et al. (1999) used solid-state NMR to demonstrate that the Y21M mutant of Ff forms better-ordered samples than wild-type, confirming the observation (Marvin et al., 1994) from X-ray fibre diffraction. Because the Y21M mutant is more rigid, the NMR spectra are sharper because the signal observed corresponds to an amide backbone in a well defined conformation (Straus, 2004 and references therein). The study of uniformly 15N-labelled samples became more tractable following a number of improvements in NMR techniques, for example higher magnetic fields, better probes, and better sample preparation techniques. Zeri et al. (2003), rather than using multiple 13C and 15N labelled samples to study the structure of the coat protein in fd phage, used one fully 15N-labelled sample and the following selectively 15N-labelled samples: Asp (D12), Leu (L14, L41), Val (V29, V30, V31, V33), Tyr (Y24), Phe (F11, F42, F45), Met (M21, M28) and Lys (K8, K40, K43, K44, and K48). These eight samples were sufficient to determine the full three-dimensional structure of the fd p8 subunit. Assignment of all of the resonances in the PISEMA spectra was made using the PISA wheel approach, developed in parallel by Marassi and Opella (2000) and Wang et al. (2000) (see Opella and Marassi (2004) and Section S2 of

9

the Supplementary material for more details). The PISEMA spectrum for the fully labelled sample is quite crowded, but by using the position of the selectively labelled amides and assuming that p8 is close to an ideal a-helical structure, the other resonances could be assigned. The chemical shift and dipolar values for Leu41, Phe42, and Phe45 in the 1985 study are compared with those in the 2003 study in Table 3. The uncertainty in the chemical shift values are reported to be 1 ppm (Cross and Opella, 1985), and the uncertainty in dipolar splitting are reported to be 500 Hz (Cross and Opella, 1985) and 100 Hz (Zeri et al., 2003). Despite the fact that the differences between the older and newer data for Leu41, Phe42, and Phe45 are larger than the reported uncertainties, the corresponding orientations of the peptide planes with respect to the external magnetic field that are derived from these parameters (see Supplementary material) only differ by 1e2 . In other words, these experimental differences imply minimal conformational differences, and thereby demonstrate the robustness of the model. The fd subunit structure that emerged from the NMR work of Zeri et al. (2003), PDB ID: 1NH4, consists of three a-helical segments (four if one includes the ill-defined residues 1e6), separated by two breaks in the dipolar waves, between residues 20 and 21 and residues 38 and 39. It was hypothesized that in the membranebound form (Section 3.1.2.1 and Almeida and Opella, 1997; Marassi and Opella, 2003), residues 7e20 are on the surface of the membrane and residues 21e38 span the membrane bilayer. But as Marvin et al. (2006) discuss, it is possible to find a continuous ahelical model which fits these data equally well, namely PDB ID: 2C0X. This model is very similar to PDB ID: 2C0W based only on the X-ray fibre data. As Zeri et al. (2003) point out, the model 1NH4 subunit helix has a periodicity of 3.68 residues per turn, whereas the subunit helix in the fibre diffraction structure PDB ID: 1IFI has 3.55 residues per turn. The geometry of the peptide backbone, especially the u torsion angle relating to the planarity of the peptide unit, can have a significant impact on the resulting structure (discussed further in Section 2.3). The current consensus model of the fd protein coat is shown in Fig. 6. The two ends of the fd phage capsid model have significantly different structures (Marvin, 1978; Marvin et al., 1992; Makowski, 1992). If the symmetry relation between p8 subunits is the same at the ends as along the shaft, the end towards which the N-terminus of the subunits points has a flared cup-shaped form, whereas

Table 3 Comparison of NMR measurements on fd phage. Reference

Residue number Leu41

Crossa Zerib

Phe42

Phe45

CS (ppm)

Dipolar (kHz)

CS (ppm)

Dipolar (kHz)

CS (ppm)

Dipolar (kHz)

217 216.6

15.3 13.8

229 229.0

18.4 19.0

214 215.7

14.4 16.0

a Cross and Opella (1985). These measurements were re-referenced to permit direct comparison of the two studies. b Zeri et al. (2003).

Fig. 6. Arrangement of subunits in the fd model. Virion axis is vertical. Axial slab, corresponding to about 1.4% of the total length of the virion. Each subunit is represented as a red space-filling coil following the protein backbone at 5 A radius. Three adjacent subunits are shown in atomic detail (yellow lines) within “semi-transparent” coils, and a single isolated subunit is displayed at the right. The coordinates of a single subunit are deposited as PDB ID: 2C0X. An electron micrograph of a full-length phage, aligned by flow on the electron microscope grid as described by Marvin and Hoffmann-Berling (1963b), is shown at the left. (Reproduced from the cover of Journal of Molecular Biology 355(2), 13 January 2006).


10


Fig. 7. Ends of the fd virion. Each subunit is represented schematically by a stack of disks 9 A in diameter and 1.5 A thick. Symmetry-related copies of the subunit were generated using the virion helix parameters. View parallel to the virion helix axis. (a) View of the flared or cup-shaped N-terminal end. (b) View of the pointed C-terminal end. Stereo pairs. From Marvin et al. (1994).

higher temperature forms is always discrete (Marvin et al., 1981). The altered symmetry means that layer lines are better resolved for the Pf1L form, and this form has therefore been analysed in most detail.

2.2. Class II phage

220 200 Chemical shift (ppm)

The biology of fd and the other Ff phages is much better understood than the biology of the Class II strains such as Pf1, but the structure of fd is less accessible to study by fibre diffraction than the structure of Pf1. Two distinct types of X-ray fibre patterns have been observed for Pf1, depending on the temperature, as outlined in Section 1.2. Below about 283 K one observes the lower-temperature pattern Pf1L, with helix parameters unit twist T and unit height H relating one subunit to the next in the helix (TL, HL) ¼ (65.915 , 3.05 A), or a helix symmetry of 5.46 units/turn. The measured axial repeat is c ¼ 214.5 A. Above about 283 K one observes the highertemperature pattern Pf1H, with (TH, HH) ¼ (66.667, 2.90 A), or a helix symmetry of 5.40 units/turn. The measured axial repeat is c ¼ 75.8 A. The axial repeat (and therefore the unit height H) can change in both forms by a few percent depending on fibre hydration and other parameters, without change in the unit twist or the helix symmetry. Slightly different unit twists (and therefore different helix symmetries) are also observed with slightly different experimental parameters, but the transition between the lower and

240

180 160 140 120 12 10 8 6 4 2 0

100 80 60

5

10

15

20

25

30

35

40

Dipolar coupling (kHz)

the other end has a pointed form (Fig. 7). The molecular details of assembly through the membrane, and the mechanism for capping the two ends with minor proteins, therefore depend on the orientation of the phage during extrusion, and this question is discussed further in Section 3.1.1.2.

45

Residue number Fig. 8. Observed chemical shift and dipolar coupling data as a function of residue number for Pf1H and Pf1L. The PISEMA data are from Thiriot et al. (2004, 2005). Pf1H : dipolar coupling (C); chemical shift (x). Pf1L: dipolar coupling (þ); chemical shift (*). From Fig. 1 of Straus et al. (2011), with permission.



The NMR PISEMA spectra of Pf1, in both the higher and lower temperature forms, are much better resolved than for fd (Thiriot et al., 2004, 2005; Opella et al., 2008). This enhanced resolution is consistent with the fact observed in fibres that it is easier to align Pf1 than fd, since more ordered samples give rise to higher resolution NMR spectra (Straus, 2004 and references therein). Based on the PISEMA spectra and dipolar waves of 15N-labelled Pf1 above and below the transition temperature, Thiriot et al. (2005) suggested that the structure of the individual subunits undergoes no significant change as a function of temperature, confirming previous X-ray fibre diffraction studies indicating that it is the orientation and packing of the subunits, not the structure of the individual subunits, that is changed at the transition temperature. Opella and co-workers suggest that the subunit comprises three distinct ahelix segments, rather than being a single continuous a-helix as in the models based on X-ray fibre diffraction data. However, analogous to the fd p8 case discussed in Section 2.1, the NMR data were shown by Straus et al. (2011) to be consistent with a continuous unkinked (or unsegmented) a-helix from residue 7 to 43. Plotting the dipolar and chemical shift data for Pf1H and Pf1L measured by Thiriot et al. (2004, 2005) as a function of residue number (Fig. 8) shows that both the dipolar and chemical shift waves follow a sinusoidal curve of frequency 3.6 residues and uniform amplitude (within experimental error) across residues 7e 43. In another approach to the same question, an ideal helix separated into segments of appropriate length was superimposed on the three segments of Pf1H NMR model 1PJF, and the same procedure was followed for the single segment of Pf1H X-ray model 1QL1. The dipolar splitting and chemical shift were then calculated, and the results are shown in Fig. 9. The waves for both models fit the data equally well, indicating that the data cannot distinguish a kinked from a continuous structure. There is therefore no requirement for segmented helices in either Pf1H or Pf1L, or for significant structural change of the subunit as a result of the transition between the two forms. The transition requires only a slight

Fig. 9. Chemical shift and dipolar coupling data as a function of residue number for Pf1H. Solid curves are the observed chemical shift and dipolar coupling values for Pf1H, from Fig. 1 of Straus et al. (2011) (see also Fig. 8). Red symbols are calculated values for model 1QL1 (continuous helix) and blue symbols are calculated values for model 1PJF (discontinuous helix). Wire representations of these two models are shown at the top, roughly corresponding to the residue numbers at the bottom. The calculated values fit the observed equally well for a continuous helix model and for a discontinuous helix model.

11

reorientation of the subunit, as discussed further in Sections 2.2.2 and 2.2.5. The segmented helix model for Pf1H is also not supported by magic angle sample spinning data. Using experimentally determined chemical shift values and secondary structure prediction programs, Goldbourt et al. (2007) present a model where residues 2e5 are in a non-helical conformation while residues 6e46 form a continuous helix. This model is also consistent with Pf1L model PDB ID: 2XKM (Straus et al., 2011). The other strains of Class II phage that have been examined (Section 2.2.4) show only the symmetry observed for the highertemperature form of Pf1. 2.2.1. The Pf1L model The Pf1L molecular model was initially refined to give a fully ahelix model, PDB ID: 1IFM, which is the Pf1L model of Marvin (1990), called “model A” by Marvin et al. (1992). This model was further refined by simulated annealing (Gonzalez et al., 1994, 1995) in parallel as a fully a-helix model (PDB ID: 2IFM), and as a model with the N-terminal five residues unwound into an extended chain, to explain the relatively ill-defined electron density near the outer radius of the virion (PDB ID: 4IFM). Magic angle solid-state NMR spin diffusion experiments on Pf1L (Goldbourt et al., 2010) are generally consistent with the sidechain interactions between neighbouring subunits predicted for PDB ID: 4IFM. Model 4IFM is preferred to model 2IFM on the basis of electron density maps and other grounds, as discussed by Gonzalez et al. (1994, 1995). PDB ID: 4IFM was further refined with respect to both X-ray and NMR data by Straus et al. (2011) to give PDB ID: 2XKM, which differs only slightly from model 4IFM (see Table 1 and Fig. 3 of Straus et al., 2011). This consensus model for the protein shell of Pf1L, based on detailed fibre diffraction and NMR data, is shown in Fig. 10. A somewhat different model (PDB ID: 1PFI) for Pf1L protein was proposed by Liu and Day (1994), based on the preliminary electron density maps of Bryan et al. (1983) and Marvin et al. (1987), and including some regions of 310 helix rather than a-helix in the subunit. Welsh et al. (2000) have shown that there are serious problems with this model: the energy of non-bonded contacts between neighbouring protein subunits is very high (that is, parts of neighbouring subunits in the model occupy the same space, which is impossible). Welsh et al. (2000) investigated this type of model further using several different methods to attempt to refine model 1PFI against stereochemical constraints and quantitative Xray data. They found that model 4IFM is clearly preferable to 1PFI. The postulated regions of 310 helix in model 1PFI have been shown experimentally by MAS NMR studies to be in fact a-helical (Goldbourt et al., 2007). Despite these difficulties, model 1PFI is still used to interpret experimental results (Lorieau et al., 2008; Goldbourt et al., 2010; Tsuboi et al., 2010; Sergeyev et al., 2011), even though more highly refined models of the Pf1L protein shell are available (Gonzalez et al., 1995; Straus et al., 2011). As is well understood for the inductive logic of experimental science, experiments such as X-ray fibre diffraction and solid-state NMR cannot unequivocally prove the validity of a specific molecular model, but they can be used to disprove a model. 2.2.2. The Pf1H model The protein subunits in the Pf1 virion are closely packed, with sidechains of each subunit interlocked with the sidechains of its neighbours (Fig. 10). This interlocking is illustrated in detail for Pf1L model 1IFM (“model A”) in Fig. 3 of Marvin et al. (1992). The change in subunit shape caused by the transition from Pf1L to Pf1H is constrained by the change in the virion helix parameters, unit twist T and unit height H, which can be measured to 0.1% accuracy. The cylindrical polar atomic coordinates (4H, zH) of an initial model for


12


Fig. 10. The refined model of the capsid of bacteriophage Pf1L. Heavy lines join Ca atoms and lighter lines join the non-hydrogen atoms of the side-chains. Charged Nþ atoms are shown blue and charged O atoms are shown red. (Stereo pairs). (a) Nearest-neighbour interactions between subunits. An arbitrary subunit k ¼ 0 and its nearest neighbours k ¼ 6 and k ¼ 11 in the virion helix are indicated. View from outside the capsid towards the virion axis (shown as a bold vertical line). (b) A 16 A thick slab of the capsid, viewed parallel to the virion axis. The subunit coordinates are deposited as PDB ID: 2XKM. Reproduced from Straus et al. (2011), with permission.

Pf1H can be derived from the corresponding coordinates (4L, zL) of Pf1L by the equations (Marvin et al., 1992)

4H ¼ 4L þ zL T H T L =H L

(1a)

zH ¼ zL HH =H L

(1b)

The coordinates of two slightly different early Pf1H models are on deposit in the Protein Data Bank. PDB ID: 1IFN is the Pf1H model described by Marvin (1990), derived from the Pf1L model 1IFM. PDB ID: 2IFN (Gonzalez et al., 1995) is the Pf1H model derived from Pf1L model 4IFM. Models 1IFN and 2IFN are similar except that the first five residues near the N-terminus are a-helix in 1IFN but extended chain in 2IFN, as in the Pf1L model 4IFM. The orientation of Tyr25 is also different for these two models, but this has no structural significance and simply reflects the absence of restraints on the outside of the virion. Welsh et al. (2000) chose model 2IFN for further study because it is most closely related to the refined Pf1L

model 4IFM, and there is no reason to suppose that the Pf1H to Pf1L transition would lead to an unwinding of the N-terminal residues. The transition from Pf1L to Pf1H is almost a rigid-body rotation of the subunit around a radius through its centre, but there is also a slight change in shape. Welsh et al. (2000) mimicked this “quasirigid-body” motion during refinement by restraining the (4, j), and c1 torsion angles of the Pf1H models to be within the same rotamers as in the Pf1L models. Welsh et al. (2000) refined model 2IFN with respect to the observed data from 12 A to 3 A resolution. The progress of this refinement was similar to that found for Pf3 (Welsh et al., 1998b). Welsh et al. (2000) then used the data from 50 A to 3 A resolution. Using the non-equatorial data in the refinement causes a shift in position of the whole subunit; adding the equatorial data causes a small change in the conformation of the sidechains near the DNA core. Refining group temperature factors starting from a constant B ¼ 10 Å2 led to values comparable to those for the Pf1L model 4IFM. Since the Pf1L data are more extensive, in refinement of the Pf1H models, Welsh et al. (2000) used the non-hydrogen atomic



13

Fig. 11. Comparison of the Pf1H and the “amalgamated” Pf1L diffraction amplitudes. Continuous curves, amplitudes of Pf1H; broken curves, amalgamated amplitudes of Pf1L. The number to the left of each curve is the layer-line index l. Scale divisions along the horizontal R axes are at intervals of 0.025 A1 from R ¼ 0.0 A1 at the left-hand side of each curve. The correlation coefficient between the two data sets shown (excluding data between R ¼ 0.0 A1 and 0.025 A1 on l ¼ 0) is 0.90. Reproduced from Welsh et al. (2000).

temperature factors taken from the Pf1L model 4IFM. The Pf1H model with the lowest overall R-value has an rmsd with respect to the starting model 2IFN of 1.6 A over all non-hydrogen atoms and 1.3 A over the backbone atoms. Validation of the refined model by

Fig. 12. Grouping of subunits in the perturbed Pf1H helix 1QL2. View perpendicular to the virion axis, N-termini of the subunits towards the top. Each subunit of index k is represented as a space-filling coil following the protein backbone at 5 A radius. Axial slab about 100 A long, corresponding to about 0.5% of the total length of the virion. Colour coding: red, k ¼ 0, 3, 6, 9,.; green, k ¼ 1, 4, 7, 10,.; blue, k ¼ 2, 5, 8, 11,.. Three adjacent subunits (k ¼ 0, 6, 11) are shown in atomic detail (yellow lines) within “transparent” rods. Heavy lines connect Ca atoms; lighter lines connect sidechain nonH atoms. (From Welsh et al., 2000).

calculating the a posteriori free R-value is illustrated by Welsh et al. (2000). The regions showing the largest differences between the models, mainly the larger sidechains, coincide broadly with the regions having the largest temperature factors. Properties of the refined model PDB ID: 1QL1, including the comparison of its transform with the observed diffraction data, are given by Welsh et al. (2000). 2.2.3. Evidence for grouping of subunits in Pf1H The Pf1H X-ray data has a maximum on l ¼ 16 at about R ¼ 0.045 Å1 that is not observed in the corresponding region of the amalgamated Pf1L data (Fig. 11); a corresponding maximum is also seen for Pf3. Refinement of Pf1H showed that this observed near-meridional intensity is forbidden by the simple helix parameters (Fig. 11). This led to the suggestion that the true asymmetric unit is best represented as a “group of three” polypeptide chains having slightly different orientations, rather than a single polypeptide chain (Welsh et al., 2000). Fig. 12 illustrates how three slightly different subunits can be grouped to give a helix with c ¼ 78.3 A but with 9 units in 5 turns instead of 27 units in 5 turns. The subunits in the unperturbed helix related by the helix parameters (TH, HH) are indexed as (k ¼ 0, 1, 2, 3, .) along the basic helix. For the perturbed helix, units (k ¼ 0, 1, 2) are no longer precisely related by (TH, HH), but units (k ¼ 0, 3, 6, 9, .) are precisely related by the perturbed helix parameters (3TH, 3HH). Then in the perturbed helix, units (k ¼ 0, 3, 6, 9, .) all have the same structure and orientation; units (k ¼ 1, 4, 7, 10, .) all have the same structure and orientation; and units (k ¼ 2, 5, 8, 11, .) all have the same structure and orientation. Why should the 27 nominally identical subunits in the helix repeat be grouped in clusters? Since this happens for both Pf1H and for Pf3, which have different p8 sequences (Table 1), it must reflect a basic property of this packing geometry, not some trivial consequence of the subunit structure. Since the layer-line breadth in the “forbidden” region of l ¼ 16 is indistinguishable from the breadth in



neighbouring regions of the diffraction pattern, the coherence length of the perturbed structure is similar to that of the helix as a whole. To explore this question further, Welsh et al. (2000) refined the structure with respect to the diffraction data with the 27 units in the unit cell defined as 9 groups of 3, related by strict noncrystallographic symmetry. Within each of these 9 groups, the 3 subunits are related by non-crystallographic symmetry imposed by a harmonic restraint. For these refined models, the 3 subunits in the group have slightly different orientations. To decide to what extent this reorientation is defined by the diffraction data, Welsh et al. (2000) tried to refine models with the whole backbone fixed, or with just the Ca atoms fixed, while allowing the sidechains to move independently for the 3 subunits in the group. Refinement of this sort was not successful in generating models that fit the l ¼ 16 X-ray data; the whole subunit must be free to move. The coordinates of the refined Pf1H model with a group of 3 subunits forming the asymmetric unit of a helix with 9 units in 5 turns are deposited as PDB ID: 1QL2. The calculated transform of the refined model is generally similar to the observed diffraction data (Welsh et al., 2000). There is also weak near-meridional intensity observed on l ¼ 26 at about R ¼ 0.062 A 1 that is not permitted for a 27/5 helix with maximum radius r ¼ 35 A, since the lowest order Bessel function term on l ¼ 26 is J11. Model 1QL2 predicts intensity in this region, although the quantitative fit of the calculated to the observed transform is poor. The most convincing evidence for reorientation of subunits as the explanation of the l ¼ 16 “forbidden” intensity is the following. Welsh et al. (2000) fitted a single subunit of model 1QL1 as a rigid body separately to each of the three subunits in the asymmetric unit of model 1QL2 and calculated the transform of this new perturbed model. The fit of calculated to observed transform is essentially the same as for the original 1QL1 model for most layer lines; but unlike 1QL1, this model predicts calculated intensity on l ¼ 16 that fits well to the observed. Except for a weak (nonobserved) meridional peak on l ¼ 9, the transform of this model shows no calculated intensity attributable to the other low-order Bessel function terms predicted by a 9/5 helix but not by a 27/5 helix. Rigid-body motion of the three subunits with respect to each other, with no change of shape or sidechain conformations, is sufficient to explain the observed l ¼ 16 near-meridional intensity. Each subunit in the group of three has the same shape, but a slightly different orientation. The structurally identical subunits follow a set of 6-start helices, with pitch 156.6 A. The interactions between the identical subunits within the 6-start helices are the nearesteneighbour interactions 0e6; the main interactions between the (non-identical) adjacent 6-start helices are the nextnearest-neighbour interactions, 0e11. Some Pf1H diffraction patterns show near-meridional reflexions at orders of about 156 A (Marvin et al., 1981), consistent with sets of structurally distinct 6start helices. The Pf1 DNA model of Marvin et al. (1992) follows the 0e6 protein helices, and grouping of protein subunits might relate to DNA-protein interactions. Models 1QL1 and 1QL2 have no significant difference in quality (Welsh et al., 2000). But the perturbed model 1QL2 has slightly lower internal energy and inter-subunit energy, and slightly lower R-values. The three subunits in the perturbed model have a similar shape, but are slightly slewed away from the orientation of the unperturbed subunit. The central regions of the subunits are still approximately related by the strict 27/5 symmetry, but the ends are displaced slightly. Releasing the strict symmetry constraints relating the subunits enables them to reorient slightly to give both a better fit to the diffraction data and a lower energy. This might be expected from the fact that a group of three subunits has more independent variables than a single subunit, although the specificity of the reorientation suggests that it is structurally significant.

The suggestion of the “group of three” symmetry has been challenged both on the basis of solid-state NMR data from aligned samples (Thiriot et al., 2004) and on the basis of magic-angle spinning NMR data (Goldbourt et al., 2007). These challenges used the deposited coordinates for the “group of three” polypeptide chains, PDB ID: 1QL2, in which there are slight differences not only in the orientations of the three subunits, but also in their conformation (because of the refinement method). But Welsh et al. (2000) also reported that if the subunits in the “group of three” are replaced by subunits with identical conformation but still with slightly different orientations, the model still explains the X-ray data (Fig. 5c of Welsh et al., 2000). The technique used by Goldbourt et al. (2007) only detects differences in structure, but not in orientation, so a group of three with the same conformation for all three subunits within the group is consistent with their findings. The fact that only one set of peaks is observed in the PISEMA spectrum reported by Thiriot et al. (2004) leads these authors to suggest that three different orientations of the subunits are not possible. However, the differences in the cross-peak positions in the spectrum are less than measurement errors if the model of Fig. 5c of Welsh et al. (2000) is used to calculate a simulated spectrum (Fig. 13; Straus et al., 2011). Therefore the “group of three” model is not ruled out by the argument of Thiriot et al. (2004). It is also notable that the simulated values for Ile in Fig. 13 are close to the experimental values reported by Thiriot et al. (2004), even for Ile3, showing that the model derived from solid-state NMR is not far from the model derived from X-ray fibre diffraction, even in the Nterminal region. The discussion of the “group of three” model indirectly illustrates the extreme regularity of the subunit shape in the virion. The calculations on the “group of three” model 1QL2 by Thiriot et al. (2004) and Goldbourt et al. (2007) both found experimentally detectable differences from the observed NMR data, even though for this model the subunits in the “group of three” differ in shape by an rmsd of only a few tenths of an Ångstrom unit. As discussed above, this is a consequence of the difference in shape of the subunits in model 1QL2, not of the difference in orientation of the subunits in the virion array.

10

Dipolar coupling (kHz)

14

8

6

4

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220

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160 140 120 Chemical shift (ppm)

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Fig. 13. Simulated spectra of a selectively 15N-Ile labelled model for three Pf1H subunits that are identical to each other in conformation, but have slightly different orientations within the phage, as for the three subunits in model 1QL2. The simulated peaks are indicated by plus symbol, open triangle and asterisk; the observed data peaks (from Thiriot et al., 2004) are indicated by a large circle. The simulated data peaks for each Ile residue (Ile3, Ile12, Ile22, Ile26, Ile32, Ile39) are nearly identical within the measurement error stated by Thiriot et al. (2004). From Fig. 9 of Straus et al. (2011), with permission.



15

2.2.4. Other Class II phage Two other Class II phage have been studied by X-ray fibre diffraction. Phage Pf3, like Pf1, infects Pseudomonas aeruginosa, and gives a diffraction pattern similar to that of Pf1H, although the DNA/ protein ratio (2.4 nucleotides/protein subunit) is more like that of fd (2.4 nt/su) than Pf1 (1.0 nt/su). The X-ray data have been analysed to give a molecular model (PDB ID: 1IFP). Phage PH75 grows at 70 C in Thermus thermophilus, a thermophilic bacteria (Pederson et al., 2001; Overman et al., 2005; Yu et al., 2006), and it shows a typical Class II diffraction pattern of very high quality, extending to 2.4 A resolution in the meridional direction, which has been analysed to give a molecular model of the protein coat. No evidence of a thermal transition was found by calorimetry, even up to 130 C. Several slightly different molecular models of PH75 have been deposited in the Protein Data Bank by Pederson et al. (2001), and PDB ID: 1HGZ is the preferred model (Table 2). 2.2.5. The Pf1 structural transition As mentioned in Section 1.2, one can get very good but slightly different diffraction patterns from the lower (Pf1L) and higher (Pf1H) temperature forms of Pf1, and these two forms can be reversibly converted from one to the other by a simple temperature change (Nave et al., 1979; Marvin et al., 1981). Dynamic lightscattering studies suggest that Pf1H is more flexible than Pf1L (Sasaki and Fujime, 1987). This temperature-induced structural transition is interesting as an example of a well-defined and easily-induced transition in a macromolecular assembly (Marvin et al., 1992; Welsh et al., 2000). This structural transition has not been observed for either the Class II phage Pf3 (Welsh et al., 1998b) or the Class II phage PH75 (Pederson et al., 2001), so the transition found for Pf1 is apparently not a direct consequence of the Class II symmetry. It may be that the transition can occur in Pf1 because the lower DNA/protein ratio allows the small change in local packing of protein subunits; or because the Pf1 virion is about twice as long, and so has about twice as many subunits, as other Class II virions. Several different methods have been used to follow the change in Pf1 structure across the transition temperature (Hinz et al., 1980; Thiriot et al., 2005) and these show a fairly sharp transition, but they do not indicate whether the structure passes through a series of intermediate states. Fibres made at the transition temperature can show a mixture of the Pf1L and Pf1H diffraction patterns, but no sign of any intermediate state (Marvin et al., 1981; Welsh et al., 2000), consistent with a first order phase transition. The X-ray experiments show that the temperature-induced structural change is associated with a small change in the orientation of subunits with respect to the virion helix axis, but the shape of the subunits is otherwise unchanged (Fig. 14). Solid-state NMR experiments on the transition (Thiriot et al., 2005) are also consistent with relatively little change in the local structure of the coat protein. Goldbourt et al. (2010) used magic angle spinning solid state NMR to compare chemical shifts assigned to hydrophobic sidechains of Pf1H and Pf1L. They find evidence for only slight adjustments of interactions between sidechains of fairly rigid subunits, consistent with the differences between Pf1L model 4IFM and Pf1H model 2IFN (Fig. 14). Comparison of the refined (Straus et al., 2011) Pf1L model 2XKM with Pf1H model 1QL1 shows a similar pattern of slight adjustments of interactions between sidechains. The NMR PISEMA data, which give residue-by-residue information about the orientation of the peptide groups, are also similar for the two forms of Pf1 (Fig. 8; see Straus et al. (2011) for further discussion of the NMR data). A change in the frequency of libration of the subunit in conjunction with the “group of three” phenomenon has been

Fig. 14. Comparison of the orientation of the Pf1L subunit 4IFM (solid lines) and the Pf1H subunit 2IFN (broken lines). Model 4IFM was moved without changing shape by rotating and translating the coordinates with respect to the virion axis, to superimpose the centres of the Pf1H and Pf1L subunits. Lines connect Ca atoms; N-termini of the subunits are towards the top. Stereo view from outside the virion towards the virion axis, which is shown as a vertical line. (From Welsh et al., 2000).

proposed as the basis of the temperature-dependent structural phase transition between Pf1H and Pf1L (Marvin et al., 1992; Welsh et al., 2000). Marvin et al. (1974a) suggested that “The possibility of a transition between two states of a tubular structure composed of rodlike structure units suggests a general model for transport by tubular structures. A structural transition could be triggered at one end of the tube and propagated along its length.If this transition involves a transient change from state I to state II and then back again to state I, a travelling wave of state II would pass down the length of the tube. .A periodic wave could function in transport like peristalsis, except that the wave of altered state need not be a wave of contraction, but could be a wave of exposed charged groups passing down an uncharged structure; a wave of exposed hydrophobic groups passing down an otherwise hydrophilic structure; or a wave of more specific molecular structure”. The general significance of phase transitions in biological phenomena has been discussed by Pollack (2003) among others. There have been attempts to link the observed Pf1 structural transition to some biological function. For instance Specthrie et al. (1987) suggested that flexibility introduced by the structural transition might be a strategy for defense of the virion against shear or other environmental stress. But it is unclear how selective pressures might favour a strain of Pf1 with the transition over one without. We feel that the transition should instead be considered as simply one of the many properties of macromolecular assemblies that are interesting in their own right, and may also prove useful in some aspect of bio-nanotechnology, perhaps as phase change memory material (Caldwell et al., 2012).


16


Table 4 Information available from various experimental methods. Array of p8 coat proteins X-ray fibre diffraction patterns contain information that can give accurate values for the symmetry operations relating the p8 coat proteins in the array, and for the orientation of the subunits. NMR data contain no information about the symmetry relationships between p8 coat proteins in the array. Thumbnail showing arrangement of subunits in fd. Axial slab, virion axis vertical. Each subunit is represented as a space-filling coil. From Fig. 4. Single p8 coat protein X-ray fibre diffraction patterns contain information about the shape and orientation of p8 coat proteins in the array, indicating that the p8 protein is largely a-helix and is oriented at a slight tilt with respect to the virion axis. Refinement of a-helix models against the X-ray data gives the position, orientation, and general shape of the a-helical p8. Solid-state NMR PISEMA data can give the orientation of each peptide group in the protein, to help define the shape of the protein, and NMR MAS data can give residue-by-residue values for the Ramachandran torsion angles (4, j) in the peptide. Raman spectroscopy can give information about orientation of aromatic sidechains. Thumbnail after Fig. 3 of Straus et al., 2011. Wire model of single subunit of Pf1 PDB entry 2XKM. Virion axis shown as a vertical line. Non-bonded interactions Molecular modelling and energy minimization of models, Raman spectroscopy, and solid-state NMR can give some information about interactions between adjacent p8 proteins in the array, or between protein and DNA. Thumbnail showing intercalation between Phe sidechains on adjacent p8 subunits in the fd model. Subunit backbones shown as helical ribbons. Virion axis shown as a vertical line at the right. Compare Fig. 9(a) of Marvin et al., 1994. DNA X-ray fibre patterns and neutron scattering experiments contain information about the approximate radius of the DNA in the virion, showing that it occupies a central position, surrounded by the p8 coat protein shell. Raman spectroscopy and solid-state NMR can give some information about the conformation of nucleotides in the DNA. Molecular modelling has led to several different speculative models for the details of DNA structure in the virion. Thumbnail after Fig. 5(a) of Marvin et al. 1992. Speculative molecular model of the DNA in Pfl, showing intercalation of bases on opposite DNA strands, surrounded by the inner region of the protein coat of PDB model 1IFM. View direction parallel to virion axis, down a box 14 Å thick.

2.3. Structure refinement X-ray fibre diffraction and solid-state NMR experiments supply information about different levels of structure (Table 4), and here we discuss in more detail how these and other kinds of information have been combined to give a robust model for the filamentous phage structure. With the relatively low resolution data available from X-ray fibre diffraction, building molecular models is an intrinsic part of the structure determination. The position of the electron density ascribed to the a-helix is defined by direct interpretation of the diffraction data. The orientation of the a-helix around its own gently curved axis is defined by accessibility (or not) of sidechains to specific reagents. The conformation of the sidechains is partly defined by knowledge-based rules and partly by calculated nonbonded contacts, since adjacent subunits are in very close contact with each other. Building detailed molecular models to explain experimental data is an essential aspect of experimentation, and the tendency of some researchers to dismiss models as purely speculative should be resisted. But when proposing a model based on fibre diffraction, it is best to deposit coordinates, so that the model can be tested. Neither fibre diffraction nor solid-state NMR data gives atomic resolution information, and it is necessary to include, in the

structure analysis of filamentous phages, the stereochemical properties of amino acid residues and DNA nucleotides, derived from accurate studies of small molecular structures (Wodak et al., 2006). This knowledge includes internal stereochemistry (bond lengths, bond angles, torsion angles). It should also include nonbonded contacts between adjacent subunits in the array, which can be as close as non-bonded contacts within subunits. The radial electron density distribution of filamentous phage has a minimum near the middle of the protein shell (Wachtel et al., 1974), and this low-density belt was interpreted as arising from the hydrophobic (and therefore low electron density) sidechains in the hydrophobic central segment of a continuous p8 a-helix subunit (Marvin and Wachtel, 1976). This minimum has instead been interpreted as indicating a break in the a-helix structure of the subunit (Makowski et al., 1980; Glucksman et al., 1992). But these authors showed no molecular model, and such a break is not confirmed by refined molecular models (Gonzalez et al., 1994, 1995). It is known that the u torsion angle in the peptide group is not precisely 180 , and this fact was used when building models of the phage by Marvin (1990) and subsequently. Building models with u constrained to precisely 180 , as in some NMR studies of filamentous phage, can produce distortion in the model and hinder detailed conclusions about the structure, as discussed further in the Supplementary material.



2.3.1. Use of Xplor-NIH for combined refinement The Xplor-NIH molecular structure determination package (Schwieters et al., 2003, 2006) enables refinement of models simultaneously with respect to fibre diffraction and solid-state NMR data, while including constraints on covalent geometry and non-bonded contacts. This package builds on the X-PLOR (Brunger, 1992) refinement package (the program CNS is closely related), and includes all the features of that package. Xplor-NIH also includes many additional molecular manipulation and refinement features, notably sophisticated refinement of hydrogen bonds, refinement of torsion angles against knowledge-based information, refinement against solid-state NMR PISEMA data, and refinement against fibre diffraction data using the functionality of the program FX-PLOR (Wang and Stubbs, 1993). Each kind of data can be separately weighted within Xplor-NIH, although this weighting depends in part on trial calculations. The X-ray fibre diffraction data of filamentous phage are continuous transform data, and are discussed by Marvin et al. (1987) and Gonzalez et al. (1995). To compare observed continuous transform diffraction data with the transform predicted by calculation from a model, visual inspection of plotted data or the use of the non-equatorial correlation coefficient is preferable to the use of a pseudo-crystallographic R-value. The observed solid-state NMR PISEMA data for aligned filamentous phage are discussed, for instance, by Zeri et al. (2003) and Thiriot et al. (2004, 2005). Many factors can contribute to variation of the chemical shift (CS) tensors used in determining the structure of a protein from an aligned sample, but valid assumptions can be made to select good CS values for structure calculations (reviewed by Saitô et al., 2010). The chemical shift and dipolar values can be tested by random permutation of questionable residues, as discussed in the Supplementary material of Marvin et al. (2006). The sign of the dipolar coupling splitting obtained from PISEMA or related experiments is degenerate, but degeneracies can be resolved in many cases (see for instance Bertram et al., 2003). As a simple measure of the fit of calculated to observed data, it is useful to calculate a normalized difference in the chemical shift and a normalized difference in the dipolar coupling (Marvin et al., 2006), analogous to the penalty function used by Kim and Cross (2002) and Bertram et al. (2003), but normalized to put the CS and dipolar coupling parameters on a similar scale. 2.3.2. Additional structural constraints from other methods Spectroscopic measurements can give details about features of the phage that are not available from either X-ray fibre diffraction or solid-state NMR, in particular regarding protein sidechain or DNA conformation. On the other hand, comparing the predicted Xray diffraction pattern from a molecular model with the observed diffraction data has the advantage that the calculation is completely rigorous, whereas many spectroscopic techniques use results from simple model systems to interpret the data, adding additional uncertainty to the results, as pointed out by Fraser and Price (1952) and others. Flow linear dichroism studies (Clack and Gray, 1992) indicate that the packaged DNA in phages fd, IKe, Pf1 and Pf3 is hypochromic relative to the purified single-stranded DNA, suggesting base stacking for these phage, but with the bases tilted away from the normal to the virion axis, with a greater tilt for the Class II phage than for the Class I phage. Ultra-violet resonance-Raman (UVRR) studies of Ff (Wen et al., 1997) suggest DNA base stacking, with base planes roughly normal to the virion axis and extensive interactions between DNA bases in the virion; and suggest that the nucleosides favour a C30 - endo/anti conformation. Polarized Raman spectra of oriented Pf1 virions suggest that the DNA bases are well-ordered,

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but are oriented with the base planes close to parallel to the virion axis, unlike Ff (Tsuboi et al., 2010). Raman spectroscopy of aromatic residues in filamentous phage is consistent with structural features of models derived by other methods (Overman and Thomas, 1995; Wen et al., 1997). Polarized Raman spectra of oriented Pf1 virions suggest that the protein subunit tyrosines (Tyr25 and Tyr40) are oriented with phenoxyl rings roughly parallel to the virion axis (Tsuboi et al., 2003), as in PDB ID: 2XKM. These Tyr25 and Tyr40 orientations of Pf1 are close to those observed for Tyr21 and Tyr24 of the Ff virion (Tsuboi et al., 2001), although since both Tyr in Ff are on the outside of the phage, they are less constrained in molecular models, see for instance PDB ID: 2C0X. Tryptophan stereochemistry in filamentous phage has been studied by polarized Raman microspectroscopy (Tsuboi et al., 1996; Tsuboi, 2002) and Raman optical activity (Blanch et al., 1999, 2001) and some of these results were incorporated into models of fd (Marvin et al., 2006). UVRR results (Overman et al., 2005) from the thermophilic phage PH75 differ from those from fd, Pf1, and Pf3 in having an unusual alanine marker (898 cm1 band), which is attributed to CaH hydrogen-bond donation by subunit Ala residues. Because alanines of the PH75 subunit occur primarily within sXXXs motifs (where s is a small side chain, e.g. Gly, Ala, Ser), and because the occurrence of such motifs in a-helices is believed to thermostabilize interhelix associations (Kleiger et al., 2002; Chakrabarti and Bhattacharyya, 2007) via Ca-H/O interactions, Overman et al. (2005) propose that Ca-H/O interactions may serve as a significant source of virion thermostabilization. Raman and UVRR signatures of PH75 are also distinguished from those of fd, Pf1, and Pf3 by several marker bands that indicate hydrophilic Trp and Tyr environments, including hydrogen bonding interactions of aromatic ring substituents. These interactions are likewise proposed as contributors to the high thermostability of PH75. UVRR markers in PH75 suggest DNA base stacking and a C20 -endo/anti conformation. Mutagenesis studies suggest that small apolar residues are highly conserved in the apolar domain of p8 for both Ff (Williams et al., 1995) and IKe (Williams and Deber, 1996) as predicted by the models. Residues that are exposed on the surface of Ff models tolerate mutations to many different side chains, as predicted by the models (Petrenko et al., 2002). Within these constraints, many viable amino acid substitutions have been found, but Trp26 in Ff and the homologous Trp29 in IKe are apparently absolutely required, perhaps as interfacial anchors between the lipid carbonyl and headgroup regions of the plasma membrane (Stopar et al., 2006). Phe11, Phe42 and Phe45 are largely conserved in this mutation analysis. These three residues are also essential for a minimized coat protein molecule to assemble with high efficiency into virions (Roth et al., 2002). It was shown for fdC models that the Phe11 ring from the k ¼ 0 subunit intercalates between the Phe42 and Phe45 rings on the k ¼ 17 symmetry-related neighbouring subunit (Marvin, 1990; Marvin et al., 1994; see also Table 4). A similar interaction is maintained in fdD models. Assembly of fd through the membrane (Roth et al., 2002; Papavoine et al., 1998) may involve such intercalation. The molecular models of Ff and IKe are further supported by experiments in which viable mutations to cysteine at various sites in p8 were probed for accessibility to cysteine-specific reagents (Khan et al., 1995). Iterative helical real space reconstruction (IHRSR) of cryoelectron micrographs (cryoEM) (Egelman, 2007) can give useful structural information without the phasing issues of X-ray diffraction, and it does not require preparation of an ordered array (a crystal or a fibre). Isolated molecules seen in an electron micrograph may be more like molecules in solution. A low resolution molecular model of the fd protein capsid has been derived by IHRSR (Wang et al., 2006), but this model is significantly different from


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models derived by X-ray fibre diffraction and solid-state NMR, and has some intrinsic flaws, as discussed by Straus et al. (2008b). In particular, Wang et al. (2006) propose a break in the a-helix near the centre of the p8 subunit, but this may be an artifact of the low density sidechains in this region of the sequence, analogous to proposals based on X-ray fibre diffraction discussed in Section 2.3. It is known that “sample heterogeneity constitutes a major methodological challenge for cryo-EM” (Spahn and Penczek, 2009). The IHRSR technique involves combining many different cryo-EM images of the virion to get a composite picture of the structure, but flexible filamentous phage virions do not all have exactly the same structure, so combining images is difficult and works best with a prior knowledge of the structure. IHRSR gives a unit height of 17.4 A relating successive pentamers in fd (Wang et al., 2006), compared with 16.1 A from X-ray fibre diffraction. Changes in unit height of about 5% as a function of changes in humidity or temperature have been identified for Pf1 and fd (Marvin et al., 1974b; Nave et al., 1979; Specthrie et al., 1987; Marvin, 1990), so the divergence between the X-ray value and the IHRSR value is not far from the range found experimentally in phage fibres. If the radius of curvature of a curved region of phage is about 1000 A as seen in some electron micrographs (Wang et al., 2006), there would be about a 5% divergence between the inner and outer circumference of a curved 60 A wide phage, similar to the observed variation in unit height. Wang et al. (2006) separated images into more than one group, but it is likely that the virions in the cryo-EM experiment occupy a continuum of structures, so the assumption that all cryo-EM images can be distributed between only a few groups may distort the results. In the aligned samples used for X-ray diffraction and solidstate PISEMA NMR, the virions are arranged parallel to each other in liquid crystal arrays, which makes them more regular, and fibres showing narrow layer lines (and therefore long coherence length) are selected to measure intensity for X-ray analysis. The structures of all the virions in the array are therefore experimentally shown to be the same. Wang et al. (2006) suggest that their data indicate polymorphism between virions and even within one virion when the virions are not in regular liquid crystal arrays, and such flexibility is certainly consistent with other data for the virion structure. But this polymorphism itself raises questions about the validity of generating a single model from cryo-EM images of many different structures. Moreover, the results of Wang et al. (2006) are based on mass/length measurements calculated relative to tobacco mosaic

Fig. 15. Stereo pair showing the array of curves representing the axes of the a-helical subunits in a segment of the Pf1L virion. The N-terminus is at the upper end of each curve. The bold curves represent subunits indexed k ¼ 0 (centre), k ¼ 6 (left) and k ¼ 11 (right). Modified from Fig. 4 of Marvin et al. (1987).

virus, but tobacco mosaic virus, with a seven-fold larger mass/ length than that of fd, may not be an appropriate standard for relative mass/length, as a consequence of differences in mass loss kinetics between thick and thin filaments, as discussed by Engel (1978) and Müller and Engel (2001). These two kinds of experiment, cryoEM and X-ray fibre diffraction, give complementary information. The X-ray experiment defines a good consensus model, from which individual virions in the cryoEM experiment may deviate. But it may be misleading to attempt to combine data from different virions in the cryoEM experiment to derive a consensus model. 2.4. Geometrical properties of subunit packing As discussed in Sections 2.1 and 2.2, the structure of the p8 subunit can be closely approximated by a single gently-curved ahelix: representing the p8 subunit as several disconnected a-helix segments is not supported. It is therefore valid to approximate each p8 subunit as a single curve, representing the a-helix axis. This approximation makes it easier to visualize not only geometrical representations of the completed virion (Fig. 15), but also geometrical representations of the mechanism by which p8 subunits may assemble out of the membrane into the completed virion. We discuss two related representations: the helicoid representation and the phyllotaxis representation. 2.4.1. Helicoid representation Inspection of a helix axis representation of the capsid structure such as Fig. 15 suggests that for some purposes the structure might be more simply represented as a set of interleaved helicoids (Marvin and Wachtel, 1976; Marvin, 1978). A helicoid is the surface swept out by a curve (in our case, one helix axis representation of the protein subunit) as it winds around a straight line axis with fixed radius and constant rotation and translation. Imagine in Fig. 15 the surface swept out as the k ¼ 0 subunit moves smoothly towards the k ¼ 6 subunit, then the k ¼ 12 subunit, then the k ¼ 18 subunit, etc. We call this the 0e6 helicoid. A different but intersecting helicoid is swepth out as the k ¼ 0 subunit moves towards the k ¼ 11, 22, 33 . subunits. The helicoid is analogous to a helix, which is the curve swept out by a point that winds around a straight line with fixed radius and constant rotation and translation. (The term “helicoid” is sometimes mistakenly used in the literature when “helix” is meant.) The helicoid has the interesting property that it has the same local geometry as the catenoid. For a simple animation illustrating the deformation between the helicoid and the catenoid, see http://en.wikipedia.org/wiki/ Catenoid. This relationship between the helicoid and the catenoid suggests a model for assembly of the capsid as it is extruded from the membrane: the protein subunits might preassemble in the membrane in a structure analogous to a segment of a catenoid, which then extrudes from the membrane as a helicoid with no change in local interactions between subunits. The application of the helicoid representation to filamentous phage structure and assembly is illustrated in Fig. 16 and is discussed further by Marvin and Wachtel (1976) and Marvin (1978). 2.4.2. Phyllotaxis representation The packing of neighbouring elongated subunits in the virion is not arbitrary. It follows the strict geometrical principles of phyllotaxis (Marvin, 1978, 1989). Discussion of phyllotaxis dates back to early attempts to understand the geometrical arrangement of leaves on a plant stem (reviewed by Jean, 1995; Green, 1999; Rutishauser and Peisl, 2001), but it has a wider general application to packing of identical units (Maciá, 2006), and has even been



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Fig. 16. Helicoid model for assembly of the virion at the membrane. (a) Top view of proteins in the membrane. Large central dot represents the DNA in the virion (the two DNA strands are not distinguished); the smaller dots represent the membrane-spanning p8 molecules. The coat proteins assemble within the membrane with adjacent protein contacts identical to the contacts in the completed virion, forming a set of interleaved spirals within the membrane (five spirals are shown here, but the actual number will depend on the details of the assembly process). These spirals grow from the outer ends by addition of further p8 subunits, while extruding at the inner ends, out of the membrane onto the virion. (bed) Detailed model of how one of these spirals can be bent into a helicoid. (b) The p8 subunits surround the DNA, with the positive C-terminus nearer the DNA. The surface swept out by these subunits is here simplified to a catenoid. (ced). The catenoid can be bent into a helicoid with no local deformation of the surface, and therefore no change in the bonding between neighbouring p8 subunits. From Marvin and Wachtel (1976).

Fig. 17. Surface lattices (radial projections) of filamentous bacteriophage capsids in the Class I and Class II symmetries, typified by the fd and Pf1 strains, respectively. Equivalent points (x) in the protein subunits of a helical capsid are projected along radii onto a cylindrical surface concentric with the virion helix axis; the cylinder is then cut vertically, opened flat and viewed from the outside. This cylindrical surface, with arrows indicating the flattening of the surface, is shown to the left of the corresponding surface lattice. Solid lines on the cylinder and on the corresponding surface lattice connect successive equivalent points with indices 0, 1, 2, 3,. The broken lines on the surface lattices represent the axis of the a-helix associated with subunit 0; identical parallel lines (not shown) are associated with all other surface lattice points. The indices of subunits nearest to an arbitrary subunit with index 0 are shown. The relationships between subunits 0, 6, and 11 are shown in more detail in Fig. 21; in particular, this figure makes clearer that the axes of the ahelical subunits are more accurately represented as gentle curves, not as straight lines as in the schematic surface lattice representation. The full array of subunit axes is illustrated in Fig. 15. (After Marvin et al., 2006).


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Fig. 18. Comparison of phyllotaxis symmetry in filamentous phage and in botany. (a) Model of the protein coat of the fd virion. View direction is parallel to the virion axis, from smaller towards larger residue number in the amino-acid sequence. A full array of symmetry-related copies of the subunit was generated by the operation of the virion helix parameters. A slab about 20 A thick was cut from the array of subunits, with subunits represented schematically by a stack of disks 9 A in diameter and 1.5 A thick. The indices of the symmetry-related subunits are shown within each subunit. Stripes connect subunits with indices differing by 5. Adapted from Marvin et al. (1994). (b). Phyllotaxis in botany, a pine cone. Adapted from http://wisconsinexplorer.wordpress.com/2010/01/23/fibonacci-cone/ with numbering from Rutishauser and Peisl (2001).

applied to optimization of a heliostat field for efficient generation of solar power (Noone et al., 2012). The neighbours nearest to the subunit indexed as k ¼ 0 along the basic helix of the Pf1 virion are the subunits with indices k ¼ 1, 5, 6, 11, 17. These are the first few terms of a generalized Fibonacci sequence, for which each term is the sum of the two previous terms. With increasing index the units alternate from one side to the other of the projected a-helix axis; and they approach increasingly close to the projected axis (Fig. 17). Similar properties are observed in the arrangement of plant leaves, and this suggested that the geometry of phyllotaxis might be relevant to Inovirus. The geometrical principles of phyllotaxis (Richter and Schranner, 1978) applied to filamentous phage predict the packing of subunits in the phage (Marvin, 1989). Fig. 18 illustrates the arrangement of subunits in the virion and in a pine cone. Green (1999) pointed out that “Patterns in plant meristems can be broadly classified as whorled or spiral”. As shown in Fig. 4(a) and (b) of Green (1999), the whorled pattern, based on a rotation axis combined with a two-fold screw axis, is reminiscent of the Class I phage symmetry (which in the notation of phyllotaxis has a “divergence angle” of 36 , like some Dicotyledonous flowers); the spiral pattern, based on a simple one-start helix, is reminiscent of

the Class II phage symmetry. Green (1999) proposed that “there is a threshold value for annulus width below which whorls are favoured and above which spirals are favored”. Translated into phage terms, this seems to mean that there is a thickness of the protein shell below which Class I symmetry is favoured and above which Class II symmetry is favoured. This topic is worthy of further thought. The principles of phyllotaxis form the basis of the “flower” model of virion assembly describing how the p8 subunits might extrude from a membrane-spanning location to their location in the completed virion (Marvin, 1989). This is easiest to visualize first in reverse, as the telescopic collapse of the virion (Fig. 19) when treated with chloroform or ether (see Section 3.1.2.1). We represent each subunit by the a-helix axis. Each a-helix is roughly equidistant along its length from its nearest neighbours. The interactions between neighbouring subunits can be represented as a pseudo Lennard-Jones potential energy, with a strong short-range repulsive element and a weaker long-range attractive element. In modelling the telescopic collapse of the virion, we compress the telescope by decreasing the unit height p relating one subunit to the next highest along the virion axis. As p decreases by a small amount the indices of the units which interact with an arbitrary

Fig. 19. Flower model for assembly of the virion at the membrane. Arrangement of subunits in Pf1 illustrating a smooth conversion from subunits in the membrane to subunits in the virion. Each subunit is represented by its a-helix axis, as illustrated in Fig. 15. Subunits with position and orientation parameters taken at successive points along the assembly pathway shown in Fig. 20 are combined to illustrate how the structure changes at the transition point. The top corresponds to the right-hand end of Fig. 20 (the intact virion) and the bottom to the left-hand end (the telescoped virion). The figure illustrates either maturation (upwards) or infection (downwards). View perpendicular to the helix axis, stereo pair. (a) N-terminal end of the coat protein oriented upwards. (b) C-terminal end of the coat protein oriented upwards (after Fig. 4 of Marvin, 1989).



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decreases, so the diameter must increase, but the subunits remain equidistant from their nearest neighbours while opening out like flower petals. As p is decreased, the subunit interactions change (Fig. 20). For the intact virion the main interactions are between subunit 0 and subunits 6 and 11 (Fig. 21). The energy of the 0e6 interaction is more negative than that of 0e11 because subunit 0 is roughly parallel to subunit 6 over more of its length. The 0e11 and 0e17 interactions become the two main interactions at about p ¼ 1 A, and these are in turn replaced by 0e17 and 0e28 at about p ¼ 0.3 A. A similar progression in nearest neighbour indices to higher terms in the Fibonacci series, called “rising phyllotaxis”, is observed as a plant grows (Jean, 1995). The effect on the structure of decreasing p of the Pf1L virion from 3.05 A to 0.35 A is illustrated in Fig. 19. As p decreases further to 0.15 A the structure flares to an inner radius of about 90 A and an outer radius of about 140 A. To visualize assembly out of the membrane, just imagine this procedure in reverse. Note that Fig. 19(a), which shows the generally accepted view of the N-terminal end of the subunit being excreted first (top), must in consequence have the C-terminal ends of the subunits in the membrane clustered quite close together (bottom), a questionable arrangement for assembly. On the other hand, the orientation illustrated in Fig. 19(b), with the C-terminal ends outwards (assembly of “flipped” subunits, discussed in Section 3.1.3) allows for a wider spacing of the subunits in the membrane. Fig. 20. Molecular dynamics of the telescope effect in Pf1. The degree of telescoping is defined by the unit height h (horizontal scale); the length of the tube is h times the total number of subunits in the virion (about 7000 for Pf1). At the right-hand end of the horizontal scale is the value for the intact Pf1L virion, h ¼ 3.05 A. The solid lines and the right-hand vertical scale give the interaction energy between the 0 subunit and subunit 5 (,), 6 (B), 11 (6), 17 (>), and 28 (x). The broken lines and the left-hand vertical scale give the inner (RI) and outer (RO) radii of the a-helix axis (the inner/ outer van der Waals radii of the protein shell are a few Ångstrom units smaller/larger than this). Local irregularities in the curves reflect defects in the refinement method, not fine structure in the energy pathway. (From Marvin, 1989, Fig. 3).

unit k ¼ 0 will remain those defined by general phyllotaxis theory: the units can slide along their length but they cannot pass through one another to find a completely different set of nearest neighbours. As p decreases, the axial dimension available to one subunit

Fig. 21. Nearest-neighbour interactions between Pf1Hsubunits. Relationship between the a-helix axes of neighbouring subunits (stereo pair). View along a radius from outside the virion, towards the virion axis, shown as a vertical line. The a-helix axis of one subunit (labelled 0) is generated as a segmented helix as described by Marvin et al. (1987). Corresponding axes of nearest-neighbours in the 0e6 and 0e11 directions (labelled 6 and 11) are generated by applying the virion helix parameters. At the line segments joining the axes the local distance between the axes and the torsion angle between the axes are measured, using the convention that ‘the angle is negative if the near helix is rotated in a clockwise direction relative to the far helix’. The values from the top down are, for the 0e6 contacts, 10.1 A, 3 ; 9.9 A, 3 ; 8.7 A, þ4 ; and for the 0e11 contacts, 8.8 A, 12 ; 10.5 A, 15 . (From Marvin, 1990).

3. Assembly of filamentous bacteriophages Now that we are confident of the molecular structure of filamentous phage, we can consider in molecular detail the important question of how this structure is assembled at the bacterial membrane. To simplify the discussion of phage assembly, we group studies as in vivo or in vitro. Under in vivo we include not only studies on biologically functional phage, as customary, but also studies on components isolated from functional phage. Under in vitro, we include experiments involving attempts to reconstitute aspects of phage assembly, for instance coat proteins inserted into artificial membranes. 3.1. Assembly at the membrane: proteins 3.1.1. In vivo studies of assembly: proteins Our view of phage assembly is shaped by our knowledge of the final structure of the phage. As discussed in Section 2, the overall architecture of the phage is the same for all strains of filamentous phage so far examined. The protein coat is an overlapping interdigitated array of elongated p8 proteins that have about 50 residues, are mostly coiled into an a-helix, and form a tube surrounding the phage DNA (Fig. 4). The detailed amino acid sequences of the coat protein may differ substantially between phage strains, but the sequences of all strains show the same general pattern, with an Nterminal acidic region, a central hydrophobic region of about 20 residues, and a C-terminal basic region. The details of assembly may differ for the two slightly different symmetry classes of phage structure. There are minor phage proteins incorporated at each end of the phage that may affect the final phage structure; and there are chaperone proteins, both phage-coded and host-coded, which may also affect the structure but are not incorporated into the phage. As we attempt to understand the assembly of the phage, we distinguish between features common to all phage strains, and features that differ between the two symmetry classes that may control the formation of the two classes. 3.1.1.1. Major coat protein. Filamentous phage both enter and leave the Gram-negative bacterial cell at adhesions between the inner (or


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plasma) and outer membranes (Bayer, 1981; Lopez and Webster, 1985; Bayer and Bayer, 1986; Lazdunski, 1995; Click and Webster, 1998; Witty et al., 2002). Lipid bilayer biological membranes consist of a w30 A-thick hydrophobic region sandwiched between two hydrophilic regions (reviewed by Bloom et al., 1991; Mouritsen and Bloom, 1993; Heimburg, 2009). The 20-residue stretch of hydrophobic amino acids from position 20 to 39 in the fd p8 sequence, if coiled into an a-helix, would be roughly the correct length to span the 30 A-thick hydrophobic region of the membrane, suggesting that the p8 protein has a membrane-spanning site before assembly of the phage (Marvin and Hohn, 1969). Experiments on the relative accessibility of the N-terminus and the C-terminus of p8 support the view that the protein spans the plasma membrane with the Nterminus in the periplasm or, as designated in the rest of this review, as outward (reviewed by Wickner, 1988). The same features are found in many unrelated strains of Inovirus, and there can be little doubt that they represent a structurally significant general property. The hydrophobic transmembrane sequence of p8 is shaded in Table 1. Site-directed mutagenesis in the hydrophobic membrane-spanning region of p8 appears to have little effect on assembly (Roth et al., 2002), and it may be difficult to draw conclusions from the detailed positions of specific residues in the p8 sequence. Host membrane proteins are needed for correct insertion of p8 into the membrane in vivo (reviewed by Dalbey and Kuhn, 2004; Dalbey et al., 2011; Dalbey and Kuhn, 2012). The p8 proteins of both Ff (Class I) and Pf1 (Class II) bacteriophages are synthesized in vivo with a hydrophobic N-terminal “signal sequence” or leader peptide. The p8 is thought to bind initially to the membrane surface by interaction of its positively charged C-terminal region with the negatively charged phospholipid head groups of the membrane lipids. The p8 with the signal peptide attached, sometimes called the procoat, then translocates into the membrane, forming a hairpin structure (Fig. 22) that spans the membrane twice, in a step that is independent of the host Sec proteins but requires a membrane potential and the host YidC membrane insertase protein (Samuelson et al., 2001; Kol et al., 2008; Stiegler et al., 2011). After the procoat is inserted in the membrane, the signal peptide is cleaved off by a host enzyme, the signal peptidase, and the mature p8 protein then resides in the plasma membrane as a membranespanning protein that is largely a-helix, with the N-terminus located in the periplasm (Fig. 5). The p8 of the Class II phage Pf3 has no signal sequence that needs to be removed (Fig. 22), which makes membrane insertion conceptually simpler, although Pf3 does also require the bacterial YidC protein for correct insertion. The p8 interacts with specific transmembrane domains of the YidC protein (Klenner et al., 2008). This catalyses the integration of the hydrophobic central region of p8 into the membrane and translocation of the N-terminal

N

N

N Pf3

N procoat

mature coat

fd

Fig. 22. Insertion of coat protein into the plasma membrane. Left: The Pf3 coat protein p8 spans the membrane with an 18-residue N-terminal domain in the periplasm and an 8-residue C-terminal domain in the cytoplasm. Right: The fd procoat protein spans the membrane twice and exposes a 20-residue hydrophilic loop in the periplasm, which is cleaved to release the mature fd coat protein p8.

hydrophilic region. Binding of Pf3 coat protein to YidC induces a conformational change within YidC that alters its periplasmic domain (Winterfeld et al., 2009; Imhof et al., 2011). The p8 can insert into lipid vesicles containing the YidC protein, but not into protein-free lipid vesicles; this is true for both Pf3 (Serek et al., 2004) and Ff (Stiegler et al., 2011), but see also Section 3.1.2.1. When the charged residues within the C-region and N-region were interchanged, the membrane orientation of the inserted Pf3 coat protein was inverted (Kiefer and Kuhn, 1999). How do the p8 proteins in the membrane assemble correctly onto the growing phage coat as it is extruded through the cell wall? This must be a quite specific mechanism, since the similar phages fd and Pf1 assemble into phage structures with distinguishably different symmetries, Class I and Class II (Section 2). The symmetry may be determined by proteins of the host species (Class I phage grow in E. coli whereas Class II phage grow in other Gram-negative species); or the symmetry may be determined by phage proteins. In the latter case, the symmetry may be determined by the arrangement of the p7 and p9 minor proteins (see Section 3.1.1.2) which initiate assembly (Endemann and Model, 1995), and later p8 subunits find their way one-by-one into the correct positions by a process similar to crystal growth. Or p1 and p11 chaperone proteins may control the symmetry of assembly (these chaperone proteins are discussed further in Section 3.1.1.3). The possibility that phage symmetry might be controlled either by the p7 and p9 initiation proteins or by the p1 and p11 chaperone proteins might be tested by generating a chimera with the proteins in question from a phage with Class I symmetry, say, replacing the corresponding proteins from a phage with Class II symmetry, and determining whether the resulting phage have Class I or Class II symmetry. In vivo evidence suggests that p8 may form multimers in the membrane. Haigh and Webster (1998) introduced cysteine at single sites in p8 of Ff phage, and tried to cross-link the protein in the membrane to learn if there is a specific neighboureneighbour interaction between adjacent subunits. Nagler et al. (2007) mutated more than one p8 residue to cysteine in Ff, and cross-linked neighbouring p8 subunits in the membrane. These results may support a model for phage assembly in which p8 proteins preassemble in the membrane. Two somewhat different but related models have been suggested for pre-assembly of the p8 protein in the membrane. In the helicoid or “ribbon” model for pre-assembly (Section 2.4.1), membrane-spanning p8 a-helices pre-assemble within the plasma membrane with the same side-by-side contacts as they have in one helicoid of the completed phage, and these ribbon arrays of protein then twist out of the membrane to form the phage coat (Fig. 16). In the “flower” model (Section 2.4.2) there is also a defined geometrical relationship between the coat proteins in the membrane, and this relationship changes according to the rules of phyllotaxis as the proteins extrude out of the membrane (Fig. 19). 3.1.1.2. Minor coat proteins. As well as the major coat protein p8, present in several thousand copies that form a single layer around the DNA of the phage, there are minor proteins, present in only a few copies, which cap the two ends of the phage (Fig. 3). The p3 minor protein is a large multi-domain protein, located at the end of the phage that enters first during infection and is extruded last during assembly (Fig. 5). The primary attachment of Ff phage to the host bacterium takes place between the pili-binding (PB) domain of the phage p3 protein and the host F-pili. Pili are elongated helical arrays of protein subunits (pilin) extending out from the bacterial cell wall, which appear to resorb into the plasma membrane of the host, drawing the virion into contact with the cell (Marvin and Hohn, 1969; Jacobson, 1972; Skerker and Berg, 2001; Marvin et al., 2003; Craig et al., 2004; Fronzes et al., 2008). The growth



and retraction of bacterial pili has similarities with filamentous phage growth and infection. Pili structures differ between different bacterial hosts, so the p3 proteins of the corresponding phage must also differ. Most of the detailed studies of p3 structure have used the Ff strains of phage, which attach to F-pili. A secondary attachment of phage to the host bacterium takes place between the TolA-binding (TB) domain of the phage p3 protein and the C-terminal domain of the host TolA protein. TolA is an outer membrane protein associated with adhesions between inner and outer bacterial membranes, which have been implicated as sites through which filamentous phage enters and leaves the bacterial cell (Webster, 1991; see also Section 3.1.1.1). During phage assembly, p3 is translocated into the membrane, perhaps using the Sec system (Rapoza and Webster, 1993). The Nterminus of p3 is extruded through the plasma membrane with the help of a signal sequence which is then cleaved off by a signal peptidase, and the protein remains attached to the plasma membrane by a C-terminal transmembrane sequence, with most of the p3 in the periplasm. The p3 protein in Ff is a multi-domain protein, with the domains separated by Gly-rich regions. Reading in the N-to-C direction, the domains have been called domain N1 (68 residues), N2 (131 residues) and C (150 residues). The N1 and N2 domains have also been called D1 and D2 by some authors. We follow Jakob et al. (2012) and refer instead to the first and second (N1 and N2) p3 domains of fd as the TolA-binding and pilus-binding domains, respectively, to avoid confusion, because in IKe phage the order of these two domains along the sequence is inverted; and we abbreviate these two domains as TB and PB. Knobs seen in electron micrographs at one end of the Ff virion have been identified with p3 (Gray et al., 1981). Digestion with subtilisin removes the knobs, leaving residue 254 at the N-terminus of the undigested portion of p3, suggesting that domains TB and PB form the knob, and the C-terminus is buried within the p8 array (Stengele et al., 1990; Kremser and Rasched, 1994; Holliger and Riechmann, 1997; Riechmann and Holliger, 1997). On the other hand, epitopes can be fused to the C-terminus of p3 and are functionally displayed on the phage surface (Fuh and Sidhu, 2000). This is consistent with the observation of Anderer et al. (1967), that serological screening tests detect slightly reactive free C-terminal serine groups in fd phage (the p3 sequence ends with -Asn-Lys-Glu-Ser-COOH). The Gly-rich linker sequence of about 35 residues between TB and PB is a low complexity or intrinsically disordered region, by the missing coordinates test (Remark 465 in the PDB entry). For fd and If1, the crystal structures of this region of p3 have been determined and the coordinates deposited in the PDB (Table 2), so the missing coordinates test is well defined. There is also a Gly-rich region between PB and the C domain. The C domain of Ff p3 has in turn been subdivided into three subdomains by study of deletion mutants. Reading in the N-to-C direction, domain C1 is necessary for virion stability in detergent, domain C2 is sufficient for release of the virion from the infected bacteria, and domain M is the trans-membrane domain (Rakonjac et al., 1999). The C domain is required for two opposite functions: insertion of the virion into the membrane during infection, and excision at the termination step of assembly and secretion. A 28residue-long segment in the p3 C domain is required for phage entry but is dispensable for release from the membrane at the end of assembly. This segment, named the infection-competence segment (ICS), works only when it is on the same polypeptide chain as the N-terminal receptor-binding domains: it does not require ICS sequences in other p3 subunits. The ICS contains a predicted amphipathic a-helix and is rich in small amino acids. A three-dimensional model of the p3 C domain has been proposed by Bennett et al. (2011).

23

Structures of portions of p3 have been determined for the Class I phages fd, If1 and IKe; and also for CTX4 (Table 2). The structure of the TB þ PB domains of p3 was determined by solution NMR and Xray crystallography (Holliger and Riechmann, 1997; Lubkowski et al., 1998; Holliger et al., 1999). There are minor apparent differences between the crystal structures of p3 from M13 phage (Lubkowski et al., 1998) and from fd phage (Holliger et al., 1999), but these appear to be due to differences in experimental methods, and the fd structure (PDB ID: 2G3P) is the standard. The p3 protein of Ff phage attaches to the tip of the F-pilus via the PB domain (Gray et al., 1981; Stengele et al., 1990; Holliger and Riechmann, 1997; Holliger et al., 1999; Deng and Perham, 2002). Deletion mutants of Ff p3 carrying TB but not PB can infect bacteria without F-pili 100-fold more efficiently than does wild-type Ff. Apparently PB masks the TolA co-receptor site on TB, and attachment of wild-type PB to the pilus releases the co-receptor site on TB for binding to TolA (Riechmann and Holliger, 1997; Lubkowski et al., 1998; Holliger et al., 1999; Chatellier et al., 1999). Adsorption of the PB domain to the pilus exposes the binding site for TolA on the TB domain by cis-to-trans isomerization at Pro213 and an associated unfolding of the hinge between TB and PB (Eckert and Schmid, 2007; Jakob et al., 2010; Hoffmann-Thoms et al., 2013). Hoffmann-Thoms et al. (2013) propose that Pro213 acts “as a molecular capacitor for the transient storage of energy. In the first step of infection, energy that originates from the binding of N2 to the pilus tip is used to unfold the hinge and thus to expose the binding site for the ultimate receptor TolA. This energy remains available even when the phage leaves the pilus tip because it is stored in the trans form of Pro-213, which prevents refolding of the hinge and thus the reassembly of the domains of G3P.” The role of p3 in the initial steps of fd infection is illustrated in Fig. 23. Adsorption of Ff p3 to the pilus can be destroyed and then rebuilt by fusing a peptide to the C-terminus of pilin, which inactivates normal p3-pili adsorption, and then fusing an antibody against this peptide to p3, thereby restoring p3-pili adsorption and infection (Malmborg et al., 1997). Different virus strains adsorb to different types of pili. Ff adsorbs to F-pili but not to N-pili, whereas IKe adsorbs to N-pili but not to F-pili; but chimeras containing both Ff and IKe PB domains can attach to either kind of pilus (Marzari et al., 1997). Phenotypic mixing is found for p3 of Cf and Xf strains, which have different host specificities, suggesting that host specificity is also conferred by pili adsorption sites for these strains (Yang and Yang, 1997). If1 phage, which adsorbs to I-pili, also contains TB and PB domains in p3, with a Gly-rich intrinsically disordered linker of about 20 residues between them. The PB domain of If1 is the adsorbtion site to I-pili, but is structurally unrelated to the PB domain of fd. The TB domain of If1 has 31% sequence identity with the TB domain of fd, and, as for fd, it binds to TolA-C. The complex between TB and TolA in If1 (Table 2) shows that TB interacts with the same site on TolA as for the TB of fd. The TB of If1 is permanently accessible, unlike wild-type fd (Lorenz et al., 2011), although fd can be reengineered so that its TB is also permanently accessible (Lorenz and Schmid, 2011). The glycine-rich segments in p3 of If1 are not as clearly clustered as in the p3 of Ff, making the clear demarcation of the sequence into S-TB-LC-PB-LC-C-M more difficult to identify. (LC stands for a “low-complexity” region, used for instance by Ford et al., 2012 as a more general description of the intrinsically disordered regions than glycine-rich.) Sequences corresponding to p3 domains in fd and If1 have been identified in several other Class I phage genomes, and some of these have Gly-rich regions. The PB sequence of IKe is not homologous to that of Ff; and the order of TB and PB within p3 is reversed in IKe, but the membrane penetration domains TB of IKe and Ff are homologous, suggesting that the penetration domain is conserved


24


Fig. 23. Schematic illustration of the role of p3 in infection of E. coli by phage fd. Infection starts with the binding of the PB (N2) domain of p3 to the tip of an F pilus (left). This is followed by partial unfolding, domain disassembly, exposure of the TB (N1) domain by cis-to-trans isomerization of Pro213, and binding of the TB domain to the C-terminal domain of TolA (right). After Fig. 1(a) of Lorenz and Schmid (2011).

between bacterial strains (Marzari et al., 1997; Jakob et al., 2012). A mutation of IKe phage containing an additional Gly-rich insertion within the Gly-rich domain of p3, that increases the length of this domain from 50 to 78 amino acids, appears to convey no advantage or disadvantage to the IKe phage (Bruno and Bradbury, 1997). In phage I2-2 DNA, a contiguous sequence of 4615 nucleotides coding for p5, p7, p9, p8, p3, p6, p1 and p4 is significantly homologous to corresponding sequences in the genomes of IKe and Ff, but no homology was observed between the consecutive DNA sequence that contains the origins for viral and complementary strand replication and the DNA replication proteins p2 and p10 (Stassen et al., 1992). As with If1, the IKe and I2-2 phage have a p3 sequence which cannot be readily subdivided into the Ff domains. The CTX4 strain of Inovirus also has a p3 protein (initially called OrfU) with a domain structure similar to that of fd p3 (Heilpern and Waldor, 2003). The TB domain of p3 and the TolA-C domain of Vibrio cholerae are structurally similar to the corresponding domains of fd p3 and E. coli TolA, although there is little sequence homology; and the TB domain is permanently accessible, more like the situation in If1 than in fd (Ford et al., 2012). The details of the structural interactions between p3 and TolA are also not the same for CTX4 as for fd (Ford et al., 2012). The Class II phage Pf1 and Pf3 infect P. aeruginosa strains K and O, respectively. The structure of the TolA-C domain of P. aeruginosa is similar to that of E. coli (Witty et al., 2002). Both Pf1 and Pf3 phage have a few copies of a minor protein analogous to the p3 of Ff phage. The p3 of Pf3 phage has a predicted signal sequence, even though the p8 does not. The p3 of Pf1 interacts with the strain K type IV pilus, whereas the p3 of Pf3 interacts with the strain O RP4 conjugative pilus but not with the strain O type IV pilus (Holland et al., 2006). The Pf3 and C2 phage strains adsorb to the shafts, not the tips, of their cognate pili (Frost, 1993). Both Pf1 and Pf3 p3 have scarcely any Gly-rich domains. For B5, there is one major Glyrich domain and the transmembrane segment is slightly shifted with respect to the C-terminus. For p3 of integrative phage 4Lf there is a 22 residue N-terminal signal sequence that is present in the DNA sequence but absent in the mature p3 protein (Lin et al., 1999). The domain structures for p3 proteins in several different filamentous phage strains are described further in the UniProtKB

data base under family “Inovirus g3p”: http://www.uniprot.org/ uniprot/?query¼family%3A%22inovirusþg3p%22&sort¼score. The incorporation of p3 into the phage coat terminates the assembly process when the end of the DNA is reached. Mutations in p3 can lead to the production of “polyphage” in which termination is skipped and a further DNA molecule is incorporated, thus increasing the phage length by integral multiples of the native length (Pratt et al., 1969; Rakonjac et al., 1999). Filamentous phage are released from the bacterial membrane by a two-step “mech-anism” involving a short C-terminal fragment of p3 (Rakonjac et al., 1999). Once incorporated into the virion, p3 is required for the stability of the proximal end of the p8 array. Mutations in p3 give phage that are much less stable to organic solvent and readily convert to contracted “intermediate” forms (discussed further in Section 3.1.2.1). The p3 and p6 proteins form a stable entity in the virion, but the C-terminal end of p6 in the virion is not accessible to anti-p6 serum (Endemann and Model, 1995; Gailus and Rasched, 1994). The p6 protein is a small protein located at the same end of the phage as p3. Disruption of the phage by mild methods dissociates p8 and frees phage DNA, but leaves p3 and p6 associated with each other (Gailus and Rasched, 1994). The p7 and p9 proteins are small proteins located at the other end of Ff phage (Endemann and Model, 1995). The Pf3 phage minor proteins are similar to those of Ff, except that p7 and p9 appear to be replaced by a single protein fusion (Luiten et al., 1985). For several other phage strains, ORFs in the DNA sequence were identified as minor proteins corresponding to p3, p6, p7 or p9 of Ff solely by their size and their position in the genome. Not all of these ORFs have been directly shown to correspond with minor proteins of the virion by amino acid sequencing, but for phage Lf, the gene coding for p6 was identified directly as coding for a minor coat protein component of the phage (Liu et al., 1997). The minor coat proteins p3, p6, p7 and p9 of Ff phage are all inserted into the inner membrane before assembly (Endemann and Model, 1995; Houbiers and Hemminga, 2004). If epitopes are added to the N-terminus of p9, the modified p9 is still correctly integrated into membrane, and viable Ff phage are assembled. This fact is a distinct advantage in using p7 and p9 for phage display, as reviewed by Løset and Sandlie (2012).



25

One question that has never been directly answered is: what is the orientation of the p8 array during assembly? Does the end towards which the N-terminus of p8 is directed come out first, or the other end? Since the p8 sits in the membrane before assembly with its N-terminal region directed outwards, it is generally assumed that the N-terminal cup-shaped distal end (Figs. 3 and 7(a)) comes out first. But the p3 also sits in the membrane before assembly with its N-terminal region directed outwards. When its C-terminal end attaches to the proximal end of the phage, to complete the assembly process, one expects the N-terminal attachment region to be directed away from the body of the phage, whereas in the completed phage, the N-terminus of p3 points in the opposite direction. In order to resolve this dilemma, Rakonjac et al. (1999) proposed that the N-terminal end of p3 folds back at the proximal end of the phage. But other possible models, involving end-toend flipping of the p8 subunit in the membrane, are discussed in Section 3.1.3. 3.1.1.3. Chaperone proteins. Phage assembly is analogous in some ways to the chaperone-usher pathway of pilus assembly (Waksman and Hultgren, 2009; Busch and Waksman, 2012) and to certain bacterial secretion systems (Russel, 1998). Correct assembly of the five Ff virion proteins and the viral DNA at the cell wall (Fig. 5) requires three phage-coded proteins (p1, p4 and p11) not found in the completed virion, and at least one host protein, thioredoxin (Russel et al., 1997). Thioredoxin apparently acts in Ff assembly as a DNA-handling protein, not a redox enzyme. Thioredoxin forms part of the DNA polymerase complex in T7 phage (not a filamentous phage), where it helps to maintain close contact between the DNA and the polymerase as the reactive site moves along the DNA; some analogous operation may take place in Ff as p5 is replaced by p8 along the DNA (Russel et al., 1997). The p1 and p11 phage proteins and the thioredoxin bacterial protein are inner membrane proteins, and affect the process of assembly (Guy-Caffey et al., 1992; Rapoza and Webster, 1993, 1995).

Fig. 25. Model of the p4 multimer in the context of the outer bacterial membrane. The p4 complex is anchored in the outer membrane at the level of the C-ring while the M and N-rings are exposed to the periplasm. The three-dimensional, surface shaded density map of p4 homomultimer is shown in blue with a wedge removed to reveal the central domain that blocks the channel at the M-ring. (Reproduced from Opalka et al., 2003).

The p1 protein of Ff phage has 348 residues, including a membranespanning hydrophobic domain from residues 254 to 273. The protein spans the membrane with the 253-residue N-terminal portion in the cytoplasm and the C-terminal portion in the periplasm (the reverse of the orientation found for other phage proteins). Adjacent to the cytoplasmic side of the membrane-spanning domain is a 13residue sequence of p1 having a pattern of basic residues closely matching the pattern of basic residues near the C terminus of p8, but inverted with respect to the sequence (Rapoza and Webster, 1995; Fig. 24). Mutagenesis shows that these basic residues are

Fig. 24. Comparison of the amino acid sequences near the transmembrane regions of p1 and p8. Phage strain shown at far left. The shaded regions represent the transmembrane portions of these proteins and the unshaded regions are the proposed amphiphilic helical regions that are exposed to the cytoplasm. Positively charged amino acids are red. The numbers at the ends of each sequence refer to the position of these residues in the intact protein. These are Class I phage except for 4Lf, for which the structural class has not yet been determined. Redrawn from Rapoza and Webster (1995) with 4Lf data from Chang et al. (1998).


26


essential for Ff growth, and they are also found in IKe and other class I strains (Rapoza and Webster, 1995), and even in strain 4Lf (Fig. 24). Presumably they interact with the DNA during assembly. There is a putative ATP-binding motif in the N-terminal cytoplasmic domain of p1 in Ff, IKe and other strains. The p1 function has been implicated in the formation of the adhesion zones between inner and outer membranes where assembly takes place (Russel et al., 1997; Rapoza and Webster, 1995). A mutation in gene 1 can revert an assembly-defective mutation in the thioredoxin gene, so p1 may interact with thioredoxin during assembly. The 108-residue p11 protein is the product of an internal initiation within the p1 gene. The cytoplasmic N-terminal portion of p11 includes the pattern of basic residues adjacent to the membrane-spanning domain of p1, but not the ATP-binding motif of p1. The p1 protein of Ff cannot replace defective p1 function in IKe, but if p1 and p4 are both replaced, phage are produced (reviewed by Russel and Model, 2006). Insofar as they have been studied, Class II phage genomes include a protein homologous to p1, but it is not clear whether these proteins include the same inverted pattern of basic residues in p1 compared with p8 found for the Class I, because Class II phage tend to have only a few basic residues (not enough to form an identifiable pattern) at the C-terminus of p8. It is possible that the relationship found for p1/p8 is related to the different symmetry (and therefore different assembly mechanism) for Class I and Class II phage. The p4 protein forms a barrel-shaped oligomer of 14 subunits in the outer membrane (Fig. 5) that has been proposed as a gated pore for virus assembly (Russel et al., 1997; Linderoth et al., 1996; Daefler et al., 1997; Opalka et al., 2003). The C-terminal half of the mature 405-residue p4 protein resides in the outer membrane, and is predicted from the sequence to contain a large fraction of beta sheet, as found for many other outer membrane proteins. The 14 monomers of the p4 oligomer are arranged in a cylindrical shape about 12 nm long. Each oligomer contains three rings of density, which correspond to the N-terminal domain (N-ring), a middle domain (M-ring), and the C-terminal domain (Fig. 25). The structure contains a large central pore, ranging in diameter from a minimum of 6 nm at its most constricted point (at the N-ring) to 8.8 nm (at the M and C-rings). The p4 multimer has homologies with secretins, bacterial proteins involved in protein export (Russel, 1998; Xie and Dalbey, 2008; Rakonjac et al., 2011), and, like many of them, expression of p4 induces the phage-shock protein operon (Model et al., 1997; Jovanovic et al., 2010). The N-terminal portion of Ff p4 extends into the periplasm, and provides translocation specificity. Filamentous phage CTX4 lacks p4 and instead uses the analogous host secretin EpsD for assembly (Davis and Waldor, 2003). Phage MDA4 also lacks p4 and uses instead the secretin PilQ of the type IV pilus assembly system (Bille et al., 2005). Phage B5, which grows in Gram-positive bacteria, has no protein analogous to p4 (Chopin et al., 2002), presumably because there is no outer membrane in its host (Schneewind and Missiakas, 2012). 3.1.2. In vitro studies of assembly: proteins 3.1.2.1. Major coat protein. As discussed in Section 3.1.1, it is difficult to dissect the details of phage assembly in vivo. There have been many attempts to shed more light on the structure and assembly of p8 by using artificial membranes, although such in vitro studies raise their own problems because it is not always clear how to construct appropriate membrane-mimicking systems. Methods to study membrane proteins are reviewed, for example, by Torres et al. (2003), Opella and Marassi (2004) and Holt and Killian (2010); the structure and properties of single-span transmembrane helices are reviewed by Moore et al. (2008). Lomize

et al. (2006, 2011, 2012) have developed theoretical methods to predict the position and orientation of membrane-spanning proteins. The basis of bacterial inner membrane structure is a doublelayer or bilayer of phospholipids, with a hydrophobic central sheet sandwiched between two sheets of charged head groups. Three different kinds of artificial system have been used to mimic this structure to study membrane proteins (De Angelis et al., 2004; Prosser et al., 2006; Franzin et al., 2007; Opella et al., 2008): - micelles or vesicles, which are small spherical soluble structures, made of detergents or phospholipids, randomly oriented in solution - bilayers, made of phospholipid double-layers stacked one on another with the bilayer normal oriented parallel to the magnetic field in NMR studies - bicelles, planar soluble disk-like constructs of phospholipid bilayers, where the disks may contain holes, like Swiss cheese. They contain both long-chain phospholipids that form planar bilayers and short-chain phospholipids that cap the rim of the bilayer. The index q gives the molar ratio of long-chain to shortchain lipids. Small bicelles, with q < 0.5, reorient rapidly in solution, and are a better model of membranes than micelles. Large bicelles, with q > 2.5, can be aligned in a magnetic field with the plane of the disk parallel to the field, so the membrane normal is perpendicular to the field (Fig. 26). Addition of lanthanide ions can align large bicelles with the membrane normal parallel to the magnetic field (Prosser and Shiyanovskaya, 2001; Prosser et al., 2006). There are many types and sizes of phospholipid molecules, and the choice should be appropriate to mimic the in vivo situation. Some studies on p8 were conducted by reconstituting pure p8 with lipid mixtures of 4:1 palmitoyl-oleoyl-phosphatidylcholine/ phosphatidylglycerol (Marassi and Opella, 2003), although this composition is not necessarily a good model for Gram-negative cytoplasmic membranes. The lipid composition of the E. coli cytoplasmic membrane is 19% phosphatidylglycerol, 74% phosphatidylethanolamine, and 3% cardiolipin with predominantly unsaturated acyl chains (Burnell et al., 1980; Epand et al., 2007), and genetic modification of membrane lipid composition can affect the structure of membrane proteins (Dowhan and Bogdanov, 2011). The cell membranes of bacteria infected with filamentous phage have an altered phospholipid composition (Woolford et al., 1974; Chamberlain and Webster, 1978). The lipids used for reconstitution should give the correct thickness of the hydrophobic region to allow the natural arrangement of the hydrophobic p8 coat protein sequence that spans it, as proposed by the principle of hydrophobic matching (Bloom et al., 1991), taking into account any tilt of the p8 away from the normal to the plane of the membrane. For example, if the hydrophobic region of the membrane in vitro is too thin, the p8 may be more tilted than it is in vivo; if it is too thick, the p8 may be less tilted in vitro than it is in vivo. Changing the length of the hydrophobic amino acid sequence in p8 protein embedding in a lipid bilayer with constant thickness can also affect how the protein sits in the bilayer (Stopar et al., 2003, 2006; Vos et al., 2007; Hemminga et al., 2010; Holt and Killian, 2010). In many experiments, pure p8 proteins are mixed with pure lipids, but different authors use different protocols, and little systematic comparison of methods has been reported. The p8 protein purified from phage by phenol extraction, or detergent extraction, or detergent and chloroform extraction before reconstitution into lipids is almost certainly not in its native conformation. Experimental results that use p8 protein prepared by such methods (McDonnell et al., 1993; Van de Ven et al., 1993; Williams and



27

Fig. 26. Samples for the study of p8 protein by solid state NMR. a)ec), p8 in lipids (artificial membranes); d), p8 in the phage, without lipids. In a), bilayers consisting of a lipid and p8 mixture are deposited on a solid support, e.g. a glass slide. The bilayers align parallel to the support, and the sample is placed in a magnetic field with the membrane normal parallel to the field B0 (indicated for all panels by the arrow at the left). In b), p8 is added to bicelles, which align with the plane of the bicelle disk parallel to the magnetic field. Both the simple bicelle and the “Swiss cheese” (bicelles with holes) models are shown. In c), a sample similar to b) is prepared, but lanthanide ions (filled circles) are added, and these change the orientation of the bicelle with respect to the external magnetic field. In d), p8 in the phage is placed inside a rotor that spins about its axis, as indicated by the blue arrow; the rotor axis is aligned at an angle of 54.7 with respect to the magnetic field.

Deber, 1996; Marassi et al., 1997; Papavoine et al., 1998; Marassi and Opella, 2003; Page et al., 2007; Park et al., 2010) for reconstituting into lipids should be carefully considered (Bayer and Feigenson, 1985; Florine and Feigenson, 1987; Spruijt et al., 1989; Zhou and Cross, 2013), to ensure as far as possible that the p8 properties in vitro are similar to those in vivo. Lipid bilayers have a characteristic “melting” temperature, which depends in part on the lipid composition of the bilayer, and below this temperature the bilayer properties are different from above this temperature. This fact has seldom been considered for in vitro studies of phage assembly, but see the discussion below of chloroform treatment of Ff, especially the work of Manning and Griffith (1985). In vivo, membrane lipids have liquid properties. That is, the growth temperature is above the membrane melting temperature, so in vitro experiments below the melting temperature are unlikely to mimic the in vivo properties (Mouritsen and Bloom, 1993; Tristram-Nagle and Nagle, 2004; Heimburg, 2009; Elson et al., 2010). The Ff filamentous phages grow at 37 C in E. coli, for which the inner membrane has a melting temperature slightly below this growth temperature (Heimburg, 2009); filamentous phage PH75 (Pederson et al., 2001) grows at 70 C in Thermus thermophilus strain HB8, for which the inner membrane has a melting temperature slightly below this higher growth temperature (Oshima and Osawa, 1983; Cava et al., 2009). As discussed in Section 3.1.1.1, different phage strains may have quite different detailed p8 sequences (Table 1), and yet all appear to assemble in much the same way and require bacterial proteins in vivo for the initial insertion of p8 into the membrane. Since there is no assay for biological function for p8 in artificial membranes and since p8 is a highly-flexible single membrane-spanning (monotopic) polypeptide chain, questions have been raised about conclusions drawn from in vitro reconstitution, for instance the suggestion that the N-terminal hydrophilic region of the Pf3 sequence has a “swinging arm” conformation (Aisenbrey et al., 2006), or that reconstituted p8 of Ff or Pf1 has an “L” conformation in membranes (Vos et al., 2009). The p8 proteins of strains Ff and Pf1 are each synthesized with a signal sequence that is required for insertion of the protein into the membrane in vivo, but is then removed by a host signal peptidase (Fig. 22). The p8 of strains such as Pf3 and B5 that have no signal sequence (Section 3.1.1.1) might be simpler models for in vitro assembly. Purified systems for reconstitution of procoat into unilamellar proteoliposomes containing the YidC insertase (Serek et al., 2004; Stiegler et al., 2011) may prove valuable for structural studies.

Both solution state NMR and solid state NMR methods have been used to gain insight into the structure of p8 embedded in model membranes. Early studies of Ff p8 protein by solution state NMR involved solubilising purified p8 in detergents such as SDS (Cross and Opella, 1979, 1981; Opella et al., 1980), octyl glucoside and deoxycholate (Henry and Sykes, 1990), and extensive studies on p8 in cholate (Hagen et al., 1978, 1979; Dettman et al., 1982) and other conditions (McDonnell et al., 1993). Early solid-state NMR studies of p8 reconstituted in lipid bilayers include those by Bogusky et al. (1988) and Marassi et al. (1997). The work on Ff revealed that the phenylalanine side chains in the acidic and basic regions of the coat protein are mobile, whereas the sidechains of the aromatic residues (Tyr and Trp) in the hydrophobic core are less dynamic. Similar findings by Henry and Sykes (1990) may be related to the fact that these systems were studied in micelles. As discussed in the Supplementary material, Section S2.3, the PISEMA spectrum of aligned proteins can define the orientation with respect to the magnetic field of both the NH vector and the normal to the peptide plane, and can thereby define both the local tilt of the a-helix with respect to the magnetic field and the azimuthal orientation of the tilted a-helix about its own axis. If the solid-state NMR spectra of p8 in bicelles consist of relatively sharp well-defined lines, a significant fraction of the p8 molecules must all have the same orientation in the membrane relative to the external magnetic field. If the molecules are randomly oriented, one records a randomly oriented spectrum, sometimes called a “powder pattern” by analogy with the crystallographic diffraction pattern observed from a powder of randomly oriented microcrystals. The solid-state NMR experiments show that the membranespanning segment of p8 is tilted by about 20e30 with respect to the membrane normal, slightly more than the tilt of p8 with respect to the axis of the completed phage, for both fd (Marassi and Opella, 2003; Opella et al., 2008) and Pf1 (Park et al., 2010). More importantly, the azimuthal orientation of the membrane-spanning portion of p8 around its own tilted a-helix axis in these experiments is not the same as for p8 in the completed virion. If the tilted p8 helix in the membrane were simply translated parallel to the plane of the membrane into a virion that is assembling with its axis normal to the membrane, residues that are known to be on the outside surface of the phage would instead be found on the inside surface of p8 in the phage, contrary to experiment, as discussed by Park et al. (2010) and discussed further in Section 3.1.3. It would be of interest to repeat the solid-state NMR experiments on the procoat protein in bicelles with the signal peptide still


28


attached to p8 (following Samuelson et al., 2001; see Fig. 22), since for both Ff and Pf1 this form (not the processed p8 without the signal sequence used by Park et al., 2010) is the form which first enters the membrane bilayer in vivo. It would also be interesting to repeat the PISEMA experiments using p8 from a phage without a p8 signal sequence, such as Pf3 or PH75 or B5. The experiments of Aisenbrey et al. (2006) on reconstituted p8 from Pf3 phage are interesting, but they do not define the polarity index (see Section S2.3 in the Supplementary material). It may also be interesting to repeat these experiments under different conditions (such as lipid composition and temperature), with different protocols for preparation of p8. Another approach for in vitro reconstitution studies could be to start not from isolated p8 molecules, but from assemblies of p8 that are only partially disassembled from their state in the phage. Amako and Yasunaka (1977) published electron micrographs of Pf1 phage treated with ether, showing that the phage collapses into hollow cylinders; they also reported that fd is insensitive to ether, but sensitive to chloroform. Marvin (1978) reported that ether treatment of Pf1 gives not only hollow cylinders, but also sheets and spheres, and suggested that these structures might mimic stages in biological assembly of phage. Nave et al. (1979) studied oriented sheets of ether-treated Pf1 by X-ray diffraction. Similar spheres and hollow cylinders or “intermediate forms” made by chloroform treatment of Ff (Fig. 27) have been studied in detail (Griffith et al., 1981; Manning et al., 1981; Lopez and Webster, 1982; Manning et al., 1983; Manning and Griffith, 1985; Stopar et al., 1998). (Intermediate-forms are sometimes abbreviated as “Iforms” in the literature, but in our text we avoid this abbreviation, since it might be confused with the “I-structure” of p8 in membranes discussed in Section 3.1.3.) With certain preparation methods, minor proteins and DNA remain associated with the intermediate forms and/or the spherical forms. The minor protein p3 is located at the flared end of intermediate forms (Lopez and Webster, 1982). Some CD studies suggest that the a-helix content of p8 in the intermediate forms is lower than in the phage, and even lower in spherical forms (Manning et al., 1983), but other spectroscopic studies suggest that converting from phage to intermediate forms does not involve a substantial change in the a-helix content of p8 (Roberts and Dunker, 1993). Disaggregation of phage induced by detergents rather than chloroform has also been studied (Khan et al., 1995; Stopar et al., 1998, 2003). The fact that both Pf1 (Class II symmetry) and fd (Class I symmetry) are converted to similar hollow cylinders and spherical forms by suitable organic solvents is consistent with their similar macromolecular structure, despite their formal difference in helical symmetry. Insertion of phage protein into membranes (reviewed by Stopar et al., 2003) requires some host functions, but intermediate forms

and spherical forms may be useful models for in vitro studies. It has been suggested that membrane proteins may have some of the characteristics of “molten globules” or “intrinsically disordered proteins”, in that they have some biological relevant conformation, but not the well-defined structure of classical crystalline proteins (Bychkova et al., 1988; Ptitsyn, 1995; Wright and Dyson, 1999; Turoverov et al., 2010). This idea has been applied to intermediate forms and spherical forms, since their interiors are accessible to organic probes in a way that intact phage are not (Dunker et al.,1991). Phage that infect Gram-negative bacteria clearly must get through the outer membrane as well as the inner membrane both during infection and during assembly. There is no model membrane system which can fully mimic such a system. The cell walls of Gram-positive bacteria, on the other hand, are comparatively simple, since they only have an inner membrane and this can be accurately mimicked by a number of model membrane systems, which have been tested extensively over the years given the high interest in developing novel antibiotics (Cheng et al., 2009, 2011, 2010; Epand et al., 2007; Pan et al., 2007). The filamentous phage B5 is homologous in many respects to other filamentous phage, but it infects a Gram-positive bacteria. B5 does not have a gene homologous to gene 4 (gene 4 encodes for p4, Fig. 5), supporting the idea that it only needs phage functions for assembly at the inner membrane. B5 does, however, have homologues to the major coat protein p8, as well as the minor coat proteins p3, p6, p7 and p9. Gram-positive bacteria, like Gram-negative bacteria, do have pili (Kline et al., 2010; Spirig et al., 2011), but it is not yet clear whether or not the initial attachment site of B5 phage is the pili, as for Gramnegative phage. Like cationic antimicrobial peptides, p8 of B5 has a net positive charge. 3.1.2.2. Minor coat proteins. Compared to the major coat protein, relatively little work has been done in vitro to elucidate the structures of the minor coat proteins in the membrane. Houbiers et al. (2001) used FTIR to study synthetic M13 p9 reconstituted in lipid bilayers and suggest, based on their findings, that p9 adopts a transmembrane a-helical structure. The lack of structural data on minor coat proteins other than p3 reflects in part the fact that these proteins are expressed in vivo in small quantities. 3.1.3. Models for protein assembly Some interesting notions about phage assembly in vivo have been generated by the studies of p8 in artificial membranes (reviewed by Stopar et al., 2003; Opella et al., 2008; Vos et al., 2009; Hemminga et al., 2010). It has been proposed that “dramatic structural changes occur when the coat protein undergoes the transition from the bacterial membrane to the phage particle during assembly” (Thiriot et al., 2004). This proposal arises from

Fig. 27. Schematic illustration of chloroform/ether treatment: possible models for intermediate I-forms, spherical S-forms, and protein complexes. Addition of solvent/buffer containing chloroform (for Ff) or ether (for Pf1) to phage at 4 C causes contraction of the particle to shorter and fatter intermediate forms in which coat proteins rearrange in some way around the DNA (step 1). More extensive treatment at 20 C causes further contraction to spherical forms (step 2) in which DNA remains in contact with minor proteins. Spherical forms may be mixed with model membranes (step 4) to reconstitute p8 into a lipid environment. Alternatively, treatment of spherical forms with detergent (step 3) may allow isolation of sub-complexes of minor proteins for study.



structural studies of phage coat protein reconstituted into artificial lipid bilayers, which suggest a largely a-helical “L structure” for Ff and Pf1, with a w10-residue N-terminal a-helix outside the membrane lying parallel to the plane of the membrane followed, after a non-helix linker, by a w20-residue membrane-spanning ahelix (Papavoine et al., 1998; Marassi and Opella, 2003; Opella et al., 2008; Park et al., 2010). The phage strain Pf3 does not require a signal sequence in vivo, but NMR studies on Pf3 p8 protein reconstituted in artificial membranes also suggested an “L structure” (Aisenbrey et al., 2006). However, the relevance of the “L-structure” has been questioned on experimental grounds (Vos et al., 2009; Hemminga et al., 2010) in favour of a continuous membranespanning a-helical coat protein, or “I structure”. Nazarov et al. (2007) and Vos et al. (2007) used fluorescence resonance energy transfer to measure distances between specific sites on p8 and suggested that these measurements can be best explained if the coat protein of M13 is a single tilted a-helix when reconstituted in lipid bilayers and “does not differ much from the native a-helical structure of the protein”. In some of these studies, p8 is present in monomeric form. Since high local concentration of p8 in membrane may be important for pre-assembly of p8 before extrusion (Nagler et al., 2007), this calls into question the relevance of the system under study. As discussed in Section 3.1.2.1, many of the in vitro studies have used simplified model membranes at temperatures unlike those of in vivo assembly, and it is still not clear whether the proposed precursor “L structure” is just an artifact of the experimental methods, since it is the membrane spanning “I structure” that is incorporated into the phage (Stopar et al., 2003). Theoretical methods have been developed (Lomize et al., 2006, 2011, 2012) to predict, for a membrane protein of known threedimensional structure, the position and orientation of the protein in the membrane. The methods take account of hydrophobic, van der Waals, hydrogen-bonding, and electrostatic soluteesolvent interactions of proteins in lipid bilayers, and also account for the preferential solvation of charges and polar groups by water and the effect of hydrophobic mismatch for transmembrane proteins. These theoretical methods have proven to be accurate for proteins whose position and orientation in the membrane are known experimentally; see http://opm.phar.umich.edu/. We have used these methods to predict the position and orientation of various models of p8 in membrane (Table 5 and Fig. 28). PDB ID: 2KSJ is the structure of Pf1 p8 coat protein in the

Table 5 Calculated orientation of p8 proteins in membrane. PDB entry 2KSJ(Pf1) 1QL1(Pf1)

n (subunits) Symmetrya Thickness ( A) DG (kcal/mol) Tilt ( )

1 1 2 3 4 5 2 3 4 5 1IFP(Pf3) 1 1HGZ(PH75) 1 2C0X(fd) 1

e e 0e6

0e11

e e e

28.2 25.8 23 13

23 24 28 29

33 48 55 85

15 14.2 13.0

23 35.4 28.6

87 78 85

13 22.8 26.6 25.2

21 27.1 13.9 26.2

80 55 38 49

Orientation of the protein was determined by minimizing the transfer energy, DG, with respect to the apolar thickness of the membrane-spanned region and the tilt of the membrane-spanning helix away from normal to the membrane. Columns headed Thickness, DG and Tilt refer to a single subunit in the membrane for n ¼ 1, but to an assembly of n subunits for n > 1. The thickness, transfer free energy DG and tilt were calculated using the server http://opm.phar.umich.edu/server.php (Lomize et al., 2012). a The helicoid direction defining the assembly of n subunits (see Section 2.4.1).

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membrane determined using solid-state NMR by Park et al. (2010). The theoretical position and orientation of this protein shown in Fig. 28 corresponds to Fig. 2(a) and (b) of Park et al. (2010) showing the experimentally determined position and orientation of this protein in the membrane. The predicted position and orientation of the transmembrane helix looks remarkably similar to the experimentally determined position, supporting the view that the methods of Lomize et al. (2006, 2011, 2012) give a reliable prediction for this system. We used the same methods to predict the theoretical position of PDB ID: 1QL1 (the structure of p8 determined in the Pf1H virion) in the membrane. The orientation of the transmembrane a-helix of 1QL1 around its own axis is very similar to that shown for 2KSJ. Compare, for instance, the positions and orientations shown in Fig. 28 for Tyr25 and Tyr40 in 2KSJ and 1QL1. Table 5 suggests that the calculated position of model 1QL1 in the membrane is energetically similar to that of model 2KSJ. Thus it is energetically feasible for the “L-structure” 2KSJ in the membrane to convert to the “I-structure” 1QL1. Indeed, because of the slight curvature of the subunit 1QL1, the N-terminal region of this subunit lies roughly parallel to the membrane surface without any “kink” required to join the transmembrane region with the membrane surface region. Roughly the same predicted orientation is found for the p8 of other phage with quite different sequences (Fig. 28), for instance Pf3, which is inserted in the membrane without needing a leader sequence; fd, which forms phage with the Class I rather than Class II symmetry; and PH75 (not shown), a thermophilic Class II phage. This generalization of the observation of Park et al. (2010) shows that the p8 in the membrane is predicted to have roughly the same position and orientation in the membrane for a variety of filamentous phage, indicating an important general property of filamentous phage assembly. The N-terminal segment of 1QL1 (and also of the other p8 proteins shown) is actually closer to the membrane surface than that of 2KSJ. Just as for p8 in the phage the observed solid-state NMR data are equally consistent with a continuous helix or a segmented helix (Fig. 9), it may be that the experimental measurements of Park et al. (2010) for p8 in membrane are also consistent with a continuous a-helix, and no “dramatic structural changes” in the subunit are required during assembly. A more accurate description of the structure in the membrane might be neither “I-structure” nor “L-structure” but a banana-shaped or “(-structure”, as proposed on the basis of FRET measurements of Ff phage (Nazarov et al., 2007). It has been suggested (see Section 2.4) that the coat protein might preassemble in the membrane with adjacent subunits interacting as they do in the 0e6 or 0e11 helicoids in the completed virion. We have calculated the membrane parameters of such assemblies for increasing numbers of subunits in the assembly (Table 5). In general the calculated membrane thickness decreases but the transfer free energy also decreases (becomes more favourable) as the number of subunits increases. However these calculations could be refined; for instance the helicoid could twist slightly without altering the interactions between neighbouring subunits. A simple way to discuss the results shown in Fig. 28 is to consider the acute angle and the obtuse angle that the tilted p8 subunit makes with the normal to the membrane, which also corresponds to the axis of the completed phage (Fig. 29(a)). As Park et al. (2010) noted for Pf1 and we have found for other phage, p8 in the membrane has its minimum energy orientation when the basic residues face into the obtuse angle. This orientation seems to be a general feature of the p8 subunit, independent of the p8 sequence or the details of the final assembly. But the basic residues near the C-terminus face into the acute angle in the completed phage. To resolve this dilemma, Park et al. (2010) suggest that


30


Fig. 28. The minimum energy position and orientation for PDB models of the p8 protein embedded in the membrane, calculated by the methods of Lomize et al. (2006, 2011, 2012). The periplasm of the cell is towards the top, and the cytoplasm towards the bottom. The two parallel horizontal lines represent the boundaries of the calculated apolar membrane core (Table 5). The charged groups on p8 are represented as small spheres, blue for basic (positive) and red for acidic (negative). Tyr sidechains are shown as wire models in midnight blue. The apolar segment of p8 (Table 1) is buried in the apolar region of the membrane. The right-hand panels show the tilted orientation in the membrane, and the left-hand panels show the subunit rotated by 90 around a membrane normal through the centre of the membrane-spanning helix. The left-hand and right-hand panels for Pf1 model PDB 2KSJ correspond to Fig. 2(a) and (b), respectively, of Park et al. (2010), showing the experimentally determined position of the protein in the membrane. This theoretical position looks remarkably similar to the experimentally determined position. Subseqent pairs of panels shows the theoretical position of Pf1H model PDB 1QL1 in the membrane; the theoretical position of Pf3 model PDB 1IFP; and the theoretical position of fdD Y21M model PDB 2C0X.

during assembly of the virion the tilted p8 Pf1 protein must rotate around its own transmembrane a-helix axis by about half a turn. As discussed by Marvin et al. (1987), two tilts need to be considered when discussing the orientation of the p8 axis with respect to the phage axis: the slope, Dr/Dz and the pitch Dz/D4. Park et al. (2010) apparently consider only the slope. The particular rotation proposed by Park et al. (2010) is not the only way to obtain the required reorientation. A similar reorientation of the a-helix could instead be obtained by a rotation of the a-helix around an axis normal to its axis and in the plane of the membrane: trans-membrane “flipping” of the p8 molecule (Fig. 29). Many small (50 residues or less)

bacterial inner membrane proteins are known (Hemm et al., 2008), some of which are found in both N-terminus outward and C-terminus outward orientations (Fontaine et al., 2011). Flipping can be affected by the phospholipid composition of the membrane (Bogdanov et al., 2008). Since the p8 is placed in the membrane with the N-terminus outward, it is often assumed that the phage assembles with the flared distal end of the p8 array (the end towards which the Nterminus of p8 points) extruding first and the pointed proximal end extruding last, as illustrated in Fig. 5. But the p3 in the membrane before assembly is oriented with its free standing N-terminus pointing in the same direction as the N-terminus of p8, whereas in the completed phage, the N-terminus of p3 points in the opposite direction. Rakonjac et al. (1999) proposed, in order to resolve this dilemma, that the C2 domain of p3 flips around the p6 protein at the pointed end of the p8 array as the phage is released from the membrane, causing the N-terminal domains of p3 to point away from the pointed C-terminal end of the p8 array. There is another possible resolution of this dilemma, suggested by the fact that p3 is found at the flared end of intermediate forms generated by chloroform treatment of phage (Lopez and Webster, 1982). In Fig. 29(c), the p8 protein 2C0X of Fig. 28 is shown associated with a schematic representation of the p3 protein, with a transmembrane a-helix, shown as a rod, and an N-terminal globular domain, shown as a filled circle, pointing outwards, into the periplasm, as initially found in the membrane (Endemann and Model, 1995). Flipping of the p8p3 complex (Fig. 29(d)) would reverse the orientation of p8 assembly into the virion and permit the p3 protein to attach to the flared N-terminal end of the phage rather than to the pointed Cterminal end. Flipping across the membrane of such a large protein complex may involve a “molten-globule” state (Bychkova et al., 1988). Somewhat different hypothetical models for attachment of p3 to the cup-shaped N-terminal end have been proposed by Marvin (1998). Further detailed structural information on this interaction is needed. The theoretical methods of Lomize et al. (2006, 2011, 2012) have also been applied to filamentous phage p8 coat protein assembled into the final phage structure (http://opm.phar.umich.edu/families. php?superfamily¼73). The predicted minimum energy structure for the Class I assemblage shows the virion axis normal to the plane of the membrane, but the virion axis of the minimum energy structure for the Class II assemblage is tilted slightly with respect to the membrane normal. This can be ascribed to the fact that every five subunits in Class I phage are all at the same axial level, whereas the subunits in Class II phage are all at slightly different levels (see Section 2 and Fig. 17). If this simple diagram were valid for phage assembly, adding new subunits to the growing Class II phage might cause the assembly to precess around a membrane normal. But the involvement of chaperone proteins, and possible preassembly of p8 subunits, may make the detailed mechanism of assembly much more complicated. Association of the tilt axis of a single subunit with the tilt axis of subunits in the assembling phage may not be as simple as discussed by Park et al. (2010). Assembly of p8 across the plasma membrane depends on a membrane electrochemical gradient (Date et al., 1980) which may be coupled with oscillating changes in the p8 a-helix conformation (Marvin, 1983) and with a Brownian ratchet (Goloubinoff and De Los Rios, 2007) to drive assembly. In summary, we favour the “flower” model for assembly of the phage, based on the helicoid representation combined with phyllotaxis theory (Section 2.4), which explains how purely geometrical principles can ensure that subunits migrate from the membrane into their final positions in the phage coat. Several different filamentous phage strains with quite different coat protein sequences have structures that follow these phyllotaxis principles. It is



31

Fig. 29. Orientation of the p8 subunit of fd Y21M phage in the membrane. The periplasm of the cell is towards the top, and the cytoplasm towards the bottom. The two parallel horizontal lines represent the boundaries of the apolar membrane core. The charged groups on p8 are represented as small spheres, blue for basic (positive) and red for acidic (negative). Tyr sidechain Y24 is shown as a wire model in midnight blue. The apolar segment of p8 is buried in the apolar region of the membrane. The p3 protein is represented in (c) and (d) schematically, not to scale, as a transmembrane rod ending at the N-terminus with a globular portion. (a) Definition of the angles between the transmembrane a-helix axis (thin blue line) and the transmembrane normal (thick blue line). The p8 subunit shown is the same as the right-hand panel of 2C0X in Fig. 28. The acute angle is labelled “ac” and the obtuse angle is labelled “ob”. The direction of the transmembrane normal is the same as the direction of the phage axis if the phage assembles by extruding normal to the membrane. The p8 in the membrane has its minimum energy orientation when the basic residues face into the obtuse angle. (b) Subunit of (a) rotated by 95 around an axis through the centre of the transmembrane a-helix axis, perpendicular to the plane of the paper. This flipping of p8 gives an orientation of the p8 in membrane that corresponds to the orientation of p8 in the completed virions, with the basic residues facing into the acute angle. (c) The p3 protein (not to scale) is inserted in the membrane with the globular Nterminus outward, and is associated with p8 protein (Endemann and Model, 1995). (d) If the p3 and p8 are flipped as a unit, the C-termini of p8 and p3 point outwards, the Nterminus of p3 would be extruded last, and the C-terminal domain of p3 could be shielded within the cup-shaped end of the p8 assemblage. The p1 protein and/or other components may be involved in this flipping.

however still not clear why some phage have Class I symmetry and some Class II, although the proposal of Green (1999) for an analogous situation in phyllotaxis, discussed in Section 2.4.2, is suggestive. Genetic experiments might help to resolve this question, for instance creating chimeras with minor proteins or chaperone proteins from one class with the p8 of the other class, as suggested in Section 3.1.1.1. 3.2. Assembly at the membrane: DNA Immediately after infection, the entering Ff single-stranded phage DNA is converted to a double-stranded replicative form (RF) that multiplies by a rolling circle mechanism (reviewed by Rakonjac et al., 2011) to give several hundred intracellular copies of the RF. Thousands of copies of the phage p5 protein are then synthesized, and binding of p5 to the viral strand of replicating DNA prevents further synthesis of the complementary DNA strand, thereby switching DNA replication from RF replication to synthesis of progeny single-stranded DNA. The p5 also regulates the synthesis of other proteins by binding to mRNA. The p5 has an additional important function, to rearrange the topology of the progeny

DNA from a random coil to a linear form, suitable for assembly in the linear progeny phage. The single-stranded DNA of integrative or temperate filamentous phage may be produced by a somewhat different mechanism, from a DNA template integrated into the host chromosome (Rakonjac et al., 2011; see also Section 4). To try to understand the mechanics of the DNA during phage assembly, we consider information about the p5 replication/assembly protein, about the phage DNA in the intracellular complex with p5, about the DNA in the completed phage, and about the differences in these features between the various strains of filamentous phage, while assuming an underlying similarity between the molecular mechanisms involved in the assembly of different phage strains. 3.2.1. The p5-DNA replication/assembly complex The Ff p5 monomer has 87 amino acid residues and forms a stable dimer in solution. The structure of the p5 dimer has been studied at high resolution by X-ray crystallography and solution NMR, and the DNA-protein interactions have been extensively studied by crystallographic, spectroscopic and genetic techniques (reviewed, for example, in articles by Mou et al., 2006; Gray et al.,


32


Fig. 30. The p5 single-stranded DNA binding protein. From PDB entry 1GPV, Guan et al. (1995). Stereo pairs. (a) The p5 protein dimer, with two DNA strands in the hypothetical DNA-binding sites. The two protein chains are related by a horizontal two-fold rotation axis in the plane of the paper, through the middle of the dimer. Each chain is represented by a rainbow coloured ribbon which runs from dark blue to light blue to green for the upper chain, and from green through orange to red for the lower chain. The two DNA strands are represented by segments of pink tubes at the right of the dimer. (b) Model of a segment of the helical p5-DNA complex. The pitch of the left-handed helix is about 90 A and the diameter is about 80 A. Side view of the helix (with the helix axis represented as a vertical black line), with twelve p5 dimers and the associated DNA, completing about one and a half turns of the helix. The p5 protein is shown as 3 Å-diameter tubes following a-carbon traces of rainbow-coloured successive dimers. The two strands of DNA are represented by a red tube and a yellow tube winding through the inner part of the complex, visible near each end. The orientation of the lowest (blue) dimer in the complex is roughly the same as the dimer shown in (a). (c) Model of a segment of the helical p5-DNA complex, as (b) but viewed down the helix axis. The p5 protein is shown as a wire following the a-carbon traces of rainbow-coloured successive dimers. The two strands of DNA are represented by a red tube and a yellow tube winding through the inner part of the complex, but only the red tube is visible since the yellow tube is below it at the same radius. The three images in this figure are not at the same scale. The coordinate system defining the p5 protein dimer in entry 1GPV is set with the y axis as the two-fold rotation axis relating the two monomers of the dimer. The centre of gravity of the non-hydrogen protein atoms lies on the y axis at 29 A from the origin, and the centre of gravity of the DNA is at 17 A. Before generating the helical array of p5 dimers, the coordinates of the dimer must be rotated by 64 around the y axis, to avoid close contacts between successive dimers in the helix. Then each dimer is moved with respect to the previous dimer in the helix by a rotation of 45 around the z axis and a translation of 11.2 A parallel to the z axis. See Skinner et al. (1994), Guan et al. (1994), Folmer et al. (1994), Konings et al. (1995), Stassen et al. (1995), Olah et al. (1995) and Su et al. (1997) for further discussion of modelling the p5-DNA complex.

2008; Masso et al., 2009; see also Table 2 and Fig. 30). The binding of p5 to DNA involves both hydrophobic and electrostatic interactions. Although p5 binds preferentially to single-strand DNA, without significant sequence dependence, it can also bind to single-

strand RNA, double-strand DNA or to G-quadruplex structures (Wen and Gray, 2004; Mou et al., 2003). About three or four nucleotides are occluded by each bound p5 monomer, depending on experimental conditions. The p5 of Class II phage Pf3 has 78



residues rather than 87, and little overall sequence homology to p5 of Ff, yet both the DNA-protein interaction and the high-resolution NMR solution structure are similar to those of Ff p5 (Casadevall and Day, 1985; Powell and Gray, 1995; Folmer et al., 1997; Horstink et al., 1999; Table 2). The p5 protein structure is related to the OB (oligomer binding)-fold, a widespread single-strand-DNA-binding motif (Murzin, 1993; Arcus, 2002; Theobald et al., 2003; Theobald and Wuttke, 2005; Zen et al., 2009). The p5 monomers in many crystal forms are related by a two-fold rotation axis. Dimers bind to two anti-parallel DNA strands. The coordinates of wild-type and point mutants of p5 protein are on deposit in the Protein Data Bank (Table 2). In the Ff-infected cell, the newly-synthesised single-stranded progeny DNA is found combined with about 1500 molecules of p5 to form a flexible nucleoprotein replication/assembly complex about 80 A in diameter and 8000e9000 A long, with several hundred complexes per cell. The complex prepackages the topologically circular progeny DNA into a linear form, with two anti-parallel but not base-paired strands. The overall structure of the complex has been studied by electron microscopy, low-resolution neutron and X-ray scattering, spectroscopy, and molecular modelling (Torbet et al., 1981; Gray et al., 1982a,b; Gray, 1989; Stassen et al., 1995; Konings et al., 1995; Guan et al., 1995; Mark et al., 1995; Olah et al., 1995; Benevides et al., 1996). The complex is a lefthanded helix of pitch about 80 A, with dimers of p5 binding the anti-parallel DNA strands at a molar ratio of about four nucleotides per p5 monomer. The DNA has a mean radius in the complex of about 17 5 A, whereas the protein has a mean radius of 30 15 A (Gray et al., 1982a,b; Olah et al., 1995), but the protein helix is open, so the DNA is not enclosed. The complex is about the same length as the Ff virion, implying that the mean axial spacing between nucleotides is about the same in the complex as in the virion. A plausible molecular model of the protein portion of the p5DNA complex was built using the high-resolution crystal structure of the p5 dimer (Skinner et al., 1994) as the basis for a helical model, with the general properties of the complex described in the previous paragraph. The interactions between neighbouring dimers in this model are similar to those found between neighbouring dimers in the high-resolution crystal structure of the Y41H p5 mutant (Guan et al., 1994), strengthening the belief in the model. A model of the Pf3 p5-DNA complex has been presented by Folmer et al. (1997). Coordinates of a typical model of the Ff p5-DNA complex, including a tetranucleotide in the hypothetical DNAbinding site, has been deposited as PDB entry 1GPV (Guan et al., 1995; Fig. 30). Similar models have been discussed by others: see caption of Fig. 30. Assembly of the Ff DNA into the phage at the bacterial membrane (Webster and Cashman, 1973) starts at a double-stranded packaging sequence that is exposed at one end of the p5-DNA complex (Schaller et al., 1969; Schaller, 1979; Bauer and Smith, 1988). Reconstituting the p5-DNA complex in vitro from purified single-stranded DNA and p5 protein can give rise to branched structures (Alberts et al., 1972; Torbet et al., 1981; Olah et al., 1995) which would not be suitable for packaging into a linear virion. A hypothetical model to avoid such branched structures was proposed by Marvin and Wachtel (1976). If p5 binds to newly revealed single-stranded regions of replicating DNA as it spins off the rolling circle, with the two anti-parallel DNA strands held together by p5 dimers, a linear p5/DNA complex could be generated, as illustrated in Fig. 31. Two successive dimers in the complex, A and B, are each composed of two monomers, A1eA2 and B1eB2, that bind to antiparallel strands of DNA. The dimer binding site of monomer A1, that normally binds to the complementary site on monomer A2, is also not far from the complementary binding site on monomer B2 in models of the complex (Folmer et al., 1994; Stassen et al., 1995;

33

Fig. 31. Hypothetical molecular mechanism to elongate a p5/DNA hairpin. (a) Singlestranded DNA, replicated off a rolling circle, binds p5 protein dimers to form a hairpin. The monomers in the p5 dimers binding to antiparallel strands of DNA are indicated as A1eA2, B1eB2 and C1eC2; the DNA regions binding these monomers are indicated by corresponding lower case letters. (b) As further DNA is replicated off the rolling circle, a new monomer, D1, binds to the new region of naked DNA, d1. The dimer-binding region of D1, once it is placed in the growing complex, is close enough to the complementary dimer-binding region of C2 to enable a dimer to form between D1 and C2 (broken line). (c) Once D1 has sequestered C2, C1 becomes available, and sequesters B2. (The apparent stretch of DNA between b2 and c2 is a consequence of the two-dimensional drawing. The difference between Figures (b) and (c) in this region can be explained by a change from a helical region of DNA in (b) to a linear region in (c)). (d) B1 then sequesters A2. (e) A1 is temporarily without a partner, but a new monomer, E1, binds to the growing DNA, e1, and the partner exchange of (b), (c), (d) is repeated, creating (f) a p5/DNA complex with an additional dimer.

Guan et al., 1995). Thus one can imagine an elongating mechanism as follows. A p5 monomer binds to the newly replicated naked single-stranded DNA adjacent to monomer A1 and sequesters the A2 monomer on the opposite DNA strand. The A1 monomer thereby released sequesters the B2 monomer on the opposite strand. Successive dimers exchange partners in this fashion as illustrated in Fig. 31, and this discontinuity propagates along the replicating DNA, perhaps by a kind of Brownian ratchet as discussed in other contexts (Kolomeisky and Fisher, 2007; Goloubinoff and De Los Rios, 2007; Serwer, 2011). The complex would remain linear while elongating in synchrony with the elongating DNA, and free


34


single-stranded Ff DNA or DNA-p5 hairpins would not be present in the cell. The structure of the single-stranded DNA binding protein RPA70 (human replication protein A), which has an OB-fold similar to that of p5, has been determined both with and without bound DNA, and the results indicate substantial changes in the protein structure following DNA binding (Bochkareva et al., 2001). Insofar as these results can be extrapolated to p5, they support our elongation model for the p5/DNA complex. The p5/DNA complex of the class I phage IKe is similar in structure to that of fd, and also follows a left-handed helix (Gray, 1989). The p5 of the class I strain If1 has 95 residues and a homologous DNA-binding loop, but no intracellular pool of If1 p5DNA complexes has been found (Carne et al., 1991). Filamentous phage Cf, unlike previously studied filamentous phage, can enter a lysogenic or integrative state in which the phage genome is integrated into the host genome (Kuo et al., 1987a,b, 1991). Phage Cf DNA is transcribed from both the positive and negative strands of the DNA (Wang et al., 1999). The p5 of phage Cf has 98 residues but modelling based on sequence homology with Ff suggests a similar molecular structure, and suggests that the more rigid appearance of the Cf complex in electron micrographs may result from the extra 11 residues at the C-terminal end of the protein (Chen et al., 1996). This more rigid structure might make the Cf complex more suitable for aligning fibres for X-ray fibre diffraction, which has not yet been reported for the Ff complex. Fibre diffraction data would enable a more robust test of molecular models of the fd p5/DNA complex. The molecular properties of the Pf1 p5 protein have been studied extensively (Davis et al., 1995; Bogdarina et al., 1998; Fox et al., 1999), although the three-dimensional structure of the protein monomer has not yet been determined. The protein is considerably larger than that of Ff (144 residues compared with 87 residues), but the putative p5 DNA binding site of Pf1 (residues 15e 30) is homologous to that of Ff (residues 12e26). The N-terminal 105-residue domain forms dimers and binds DNA much as the intact protein. But in the absence of a crystal structure of the Pf1 p5 dimer, it is conceivable that the monomers in the dimer are not related by a two-fold rotation axis, as the Ff p5 structure, but instead are in a tandem orientation, like the two single-strand DNA-binding domains of RPA70 (Bochkareva et al., 2001). The Cterminal 38-residue domain, which is rich in Ala, Gln and Pro residues, is flexible even in the complex with DNA, as indicated by sharp resonances in the 1H NMR spectra, and it may be compared with “intrinsically disordered” or “molten globule” proteins (Turoverov et al., 2010). This domain increases the cooperativity of DNA binding in the nucleoprotein complex. The helical p5-DNA complex of Pf1 (Kneale et al., 1982; Gray et al., 1982a,b; Kneale and Marvin, 1983; Kneale et al., 1991) is fairly flexible, but to a first approximation the parameters (length, pitch and DNA radius) of the Pf1 complex are similar to those of the Ff complex, except that the p5-DNA helix of Pf1 is right-handed (Gray et al., 1982a,b), whereas that of Ff is left-handed. Low resolution X-ray fibre patterns have been reported for the Pf1 p5-DNA complex but not the fd-complex (Kneale et al., 1982; Kneale and Marvin, 1983), a distinction perhaps traceable to the less welldefined larger pitch of the fd complex. The radius of the DNA in the Pf1 complex is calculated to be smaller than in the fd complex if one assumes that the Pf1 DNA in the complex is circular (Gray et al., 1982a,b) but neutron scattering indicates high density, possibly due to DNA, at a larger radius in the Pf1 complex than in the fd complex (Kneale et al., 1991). A hypothetical model to explain this difference in hand has been proposed by Welsh et al. (1998a), see Section 3.2.2. If the Pf1 DNA in the p5-DNA complex is circular (see Section 3.2.2), the axial spacing from one nucleotide to the next would be

about the same in the Pf1 complex as in the Ff complex, but this is only about half the corresponding axial spacing between nucleotides in the Pf1 virion. During phage assembly the Pf1 DNA, but not the Ff DNA or the Pf3 DNA, would therefore stretch to about double the length that it has in its complex with p5. This doubling happens with Pf1 but not Pf3, both Class II phage: it is not correlated with the structural class of the phage. The p5-DNA complex of the thermophilic filamentous phage PH75 (Pederson et al., 2001) might have a more stable and rigid structure at room temperature, as found for other proteins from thermophilic bacteria (Cava et al., 2009), so the p5-DNA complex of PH75 could be analysed in more detail by fibre diffraction. 3.2.2. DNA in the virion Pf1 phage is about twice as long as fd phage, even though the size of the encapsidated DNA is similar for the two species (6408 nucleotides for fd, 7349 nucleotides for Pf1), and the length of the corresponding p5-DNA complex is about the same for the two species. Marvin and Wachtel (1976) suggested that the difference between the length of Pf1 phage and that of fd might be traced to the difference in positive charge on the inner surface of the protein capsids and a requirement to match the charge per length of the DNA to that of the protein. This proposal is supported by genetic engineering experiments on fd (Hunter et al., 1987; Greenwood et al., 1991; Symmons et al., 1995) in which changes in the number of positively charged sidechains in the C-terminal region of p8 are accompanied by changes in the length of the phage. However this proposal cannot be a full explanation, as discussed by Welsh et al. (1998b), because Pf3 phage has the same nucleotide/subunit ratio as fd (2.4 nt/su) but Pf3 has only two positive side-chains in the C-terminal region of p8, whereas fd has four. The question of the structure of the DNA in the Pf1 virion has generated some heat but not much light. Precise measurements of the DNA/protein ratio of Pf1 indicate 1.0 nt/su, leading to the suggestion that there might be a specific stoichiometric relationship between the DNA and the protein (Day et al., 1979). For circular Pf1 DNA, there must be two strands, an “up strand” and a “down strand”, packaged within the Pf1 virion, and the proposed stoichiometric relationship would require that each DNA strand follows the basic Pf1 helix, with successive nucleotides on each DNA strand interacting with alternating protein subunits along the protein helix. By simple geometry, the distance between alternating subunits can only be spanned if each sugar-phosphate chain is very near the axis of the phage, with the bases pointed outwards. This “everted DNA” model (Day et al., 1979; Liu and Day, 1994) is still discussed as a serious possibility for Pf1 DNA (Tsuboi et al., 2010; Sergeyev et al., 2011), so it may be worth repeating once again the discussion by Marvin et al. (1981) pointing out that the model of a DNA sugar-phosphate chain following the basic Pf1 protein helix is based solely on numerology. One simple test of the significance of the 1.0 nt/su numerology might be to repeat for Pf1 the experiments on fd, namely changing the number of positively charged sidechains in the C-terminal region of p8 and examining the length of progeny phage. Greenwood and Perham (1989) studied phage with altered C-terminal positive charge in Pf1 p8 grown in E. coli, and found no phage production, but this could be a consequence of not using the natural host, P. aeruginosa. Different strains of Inovirus have different nucleotide/subunit ratios; why should the fact that Pf1 has 1.0 nt/su have any special significance? The model based on this numerology is flawed because it fails to explain how each of the two oppositely-directed DNA strands (necessary for circular DNA topology) can have the same relationship with a protein helix that runs in only one direction. In each strand of the DNA, the axial distance from one nucleotide to the next along the DNA is twice the axial distance



from one protein subunit to the next, so this type of model predicts layer-lines on the fibre diffraction pattern of the virion that do not coincide with the protein layer-lines. Such layer lines have been sought but not observed (Welsh et al., 1998a), whereas if the DNA within the virion has a regular structure, one expects the corresponding regular layer lines. If the 1.0 nt/su ratio has no stoichiometric significance, it is not necessary to hypothesize everted DNA. In the everted DNA hypothesis, the negatively charged phosphate groups on the two strands would be close together at a small radius in the virion, so one must postulate some mechanism to bring these highly charged groups close together during assembly. Order parameters derived from solid-state NMR of Pf1 indicate that the basic residues of the protein near the C-terminus, which would interact specifically with the DNA in the everted DNA hypothesis, are in fact highly mobile (Lorieau et al., 2008). Model 1PFI of Liu and Day (1994) was based on a low-resolution electron density map calculated by a maximum entropy method (Bryan et al., 1983; Marvin et al., 1987), but model 1PFI goes well beyond the original interpretation of this map in attempting to deduce details of the protein and DNA, and in particular fails to address the comment by Marvin et al. (1987) that “any fine structure imposed on the central DNA core by the maximum entropy calculation would be suspect”. Flaws in the protein model 1PFI have been discussed in detail by Welsh et al. (2000) and are summarized in Section 2.2.1. Another model for the DNA in the Pf1 virion may be interesting to consider, namely that the single-stranded Pf1 DNA in the virion is linear, not circular. The DNA of parvoviruses such as minute virus of mice and adeno-associated virus, which are isometric animal viruses, is about the same size as filamentous bacteriophage DNA, but it is topologically a linear single-stranded DNA molecule, which is converted to a double-stranded RF during the first steps of infection, and then gives rise to linear progeny single strands in the progeny virions (Bourguignon et al., 1976; Astell et al., 1983; Berns, 1990). If Pf1 DNA in the virion is really a linear single-strand, not circular, then a single DNA strand could stretch from one end of the virion to the other, following the basic protein helix, so no extra layerlines would be observed on the diffraction pattern. The unusual features of the Pf1 p5-DNA interactions, such as “single-site binding” under certain conditions (Carpenter and Kneale, 1991), and also the different properties of the p5-DNA complex, as discussed in Section 3.2.1, could arise from the need to package the DNA in a different way from other filamentous phage. In fact the packaging into the p5-DNA complex would be simpler for a linear DNA: there would be no need to postulate a mechanism for the DNA to fold back on itself in the complex. The initial consensus sequence of Pf1 was not circular, although this was attributed at the time to technical difficulties (Hill et al., 1991). The Pf1 genome has several more ORFs than in Ff phage (Hill et al., 1991) and the extra proteins in Pf1 might be needed for such more complicated DNA replication. Integrative (temperate) filamentous phage such as CTX4, Ypf4, 4RSM, SMA9, Pf4 and Pf5 may also have a stage in which the phage genome is linear (Hagemann et al., 2006; Chouikha et al., 2010; Rakonjac et al., 2011; Askora et al., 2012), and it is not yet clear in such cases whether the DNA in the mature phage is linear or circular. The nucleotide base composition of the single-stranded DNA of minute virus of mice corresponds to about 1/3 T, with the A/T and G/C ratios far from 1 (Astell et al., 1983). This high-T composition is also found for the single-stranded DNA phages 4X174, fd, IKe and Pf3, and has been shown to correlate with an unusual codon usage, namely preferential use of T (U) as base 3 of the codon (Sanger, 1975; Staden and McLachlan, 1982; Luiten et al., 1985), perhaps due to altered “wobble” imposed by phage infection (Denhardt and Marvin, 1969; Agris et al., 2007), but see Cardinale et al. (2013) for an alternative view. The nucleotide composition of Pf1 single-

35

stranded DNA, unlike the DNA of other filamentous phages, corresponds roughly to A/T and G/C equal to 1, even though there is no evidence for extensive base pairing in the single-stranded Pf1 DNA. For adeno-associated virus, both the plus and minus DNA strands are packaged in separate virions (Bourguignon et al., 1976). Packaging of both strands in similar numbers might explain the observed nucleotide composition of “single-stranded” Pf1 DNA. A circular topology for the single-stranded DNA isolated from the Pf1 virion was postulated to explain the two components found by sedimentation analysis, thought to be circular and nicked circular (linear) forms (Wiseman et al., 1976), but if the plus and minus strands have slightly different solution properties, this could instead explain the two components found by sedimentation analysis. Solution studies of the DNA isolated from fd phage showed that the fd DNA is topologically a circular single strand, but this study used both sedimentation and viscosity to characterize the DNA (see Section 1.1). Circular single-stranded DNA isolated from Pf1 virions was also seen in electron micrographs (unpublished data cited by Wiseman et al., 1976). But none of the experiments proves unequivocally that the single-stranded DNA in the Pf1 virion is circular. Yet another hypothetical model for non-everted DNA in the Pf1 virion was described by Marvin et al. (1992), with bases on each of the oppositely-directed DNA strands partly intercalated between the bases on the opposing strand, as originally suggested by Marvin and Wachtel (1976). Experimental evidence (Chou and Chin, 2001) indicates that such intercalation can be found for DNA, even when Watson-Crick pairing of opposite strands would otherwise be possible. Linear dichroism spectra of phage oriented by flow indicate that the DNA in Pf1 is hypochromic relative to purified single-strand DNA, suggesting base stacking in Pf1, as in other filamentous phage, with an angle of about 50 between the phage axis and the plane of the bases (Clack and Gray, 1992), although base stacking has been questioned on the basis of UV absorbance measurements of unaligned samples of Pf1 (Kostrikis et al., 1994). Polarized Raman spectra of oriented Pf1 fibres suggest that the base planes are close to parallel to the virion axis (Tsuboi et al., 2010). The results of Chou and Chin (2001) suggest how the model of Marvin et al. (1992) might be modified to be consistent with tilted bases. In the DNA model proposed by Marvin et al. (1992), the plane defined by the bonds from phosphorus to the two nonesterified oxygens is approximately perpendicular to the virion axis, consistent with the orientation determined by 31P NMR (Cross et al., 1983; Opella et al., 2008). It might be interesting to repeat for Pf1 phage the experiments of Shen et al. (1979), who treated intact fd phage with a DNAcrosslinking reagent and then extracted the DNA and demonstrated by electron microscopy the presence of intra-strand DNA crosslinks. If only one DNA strand runs along the length of Pf1 phage, no such crosslinking should be observed. The reagent used by Shen et al. (1979) crosslinks adjacent pyrimidine bases, so everted DNA might also fail to show crosslinks with this reagent. The facts about the DNA in the p5/DNA complex and in the virion give strict constraints on the dynamics of DNA geometry as it passes from the complex with p5 to the virion. A schematic hypothetical model for the transfer of DNA from the complex to the virion to explain the opposite hands of Ff and Pf1 complexes, based on these geometric considerations, is described by Welsh et al. (1998a). In this hypothetical model, the helical structure of the DNA as it passes from the complex to the virion is altered by a general “rotator” embedded in the bacterial membrane, that is the same for Pf1 as it is for Ff and other filamentous phages. This might involve the host thioredoxin protein, which is necessary for Ff virion assembly (Russel, 1991). Specifically, the Ff assembly machinery operates on the left-handed complex to increase the (negative) unit twist between DNA phosphate groups during


36


assembly, thereby decreasing the phosphate radius to enable the DNA to be packaged in the virion. If the Pf1 rotator operates in the same negative sense on the right-handed Pf1 complex (which may have evolved to be right-handed because of the altered p5 protein), the unit twist between DNA phosphate groups will be decreased during assembly. The only way for the radius of the DNA to be decreased, to enable the DNA to be packaged in the virion while maintaining a feasible distance between phosphate groups, is for the axial spacing between the nucleotides to be increased, as observed. This model might be tested by replacing p5 in Pf1 by the corresponding protein from Pf3. This would be predicted to give a left-handed p5-DNA complex and a shorter virion. 4. Discussion and conclusions Filamentous bacteriophages are of general interest in molecular biophysics because their relatively simple physical and genetic construction enables study at a fundamental level. A simple biological system opens the way to a study of the “systems” aspect of structural molecular biology: how the interactions between the different constituents of the system are controlled at the structural level. The life-cycle of the phage is embedded in a more complicated ecosystem, the bacterial host, but to a first approximation the ecosystem can be discounted by comparing the host with and without the infecting phage (Karlsson et al., 2005). For instance, the phage-shock protein response induced by Ff infection (Brissette et al., 1990; Model et al., 1997; Rakonjac et al., 2011; Huvet et al., 2011) has been analyzed as a first step in developing explanatory and predictive mathematical models of biological systems (Toni et al., 2011). Filamentous phage have also been used to study conflict mediation systems (Sachs and Bull, 2005). One important advantage of filamentous phage over other systems, as we have reviewed here, is that several steps along the relatively simple life cycle have been examined by detailed structural studies, notably the p8 protein assembly that forms the phage coat; the p3 protein that is involved in the initial steps of infection; and the p5 replication/assembly DNA-handling protein. Some filamentous phage can integrate their genome into the host genome, and are reminiscent of the classical temperate phages like lambda; although, like other filamentous phage, in the non-temperate phase they can continuously excrete phage from the host without lysis (reviewed by Rakonjac et al., 2011; Askora et al., 2012; see also Kuo et al., 1987a,b, 1991; Hagemann et al., 2006). They can be of agricultural (Kawasaki et al., 2007) or medical interest because the phages may be involved in pathogenesis. For instance CTX4 integrative phage that infects V. cholerae bacteria carries the gene coding for cholera toxin, and can convert non-pathogenic strains of V. cholerae to pathogenic strains (Davis and Waldor, 2003). Integrative filamentous phage Ypf4 contributes to the pathogenicity of Yersina pestis, the Gramnegative bacterium that causes plague (Derbise et al., 2007; Chouikha et al., 2010). Some features of the system have also proven to be useful as technological tools. When a gene coding for a foreign protein is fused to one of the coat protein genes, a virion can be produced that expresses the foreign protein on the surface of the virion (Smith, 1985), and foreign proteins may permit binding of further nonbiological constituents. The book edited by Barbas et al. (2001) and the reviews by Kehoe and Kay (2005), Hertveldt et al. (2009), Bradbury et al. (2011), and Løset and Sandlie (2012) describe the display of foreign epitopes on the surface of filamentous bacteriophage. Some medical applications of phage display are discussed by Frenkel and Solomon (2002), Solomon (2007), Deutscher (2010) and Ghosh et al. (2012).

Applications of filamentous bacteriophage to bionanotechnology have been expanding rapidly, notably due to contributions by Angela Belcher and colleagues. Some early work is summarized by Hemminga et al. (2010) (especially Table 2 of that paper) and Rakonjac et al. (2011). Other developments have been reported by Huang et al. (2005), Chiang et al. (2007), Khalil et al. (2007), Dang et al. (2011), Lee et al. (2012), Hess et al. (2012), Murugesan et al. (2013), and Oh et al. (2013). One advantage of phage for nanotechnology is that once a genetically stable modification of the phage has been constructed, unlimited identical copies can be produced by straightforward microbiological methods. A method to create complex nanoscale shapes using singlestranded circular filamentous phage DNA as a template (“DNAorigami”) was described by Rothemund (2006) and developed by Douglas et al. (2007) to create DNA nanotubes for alignment of membrane proteins in NMR studies. The DNA origami method has been widely used in other contexts, for instance by Bai et al. (2012) to create a number of previously undescribed DNA topologies, some of which may be relevant to the structure and/or assembly of the DNA in filamentous phage. The liquid-crystal properties of filamentous phage have been extensively studied (reviewed by Zhang and Grelet, 2013; see also Barry et al., 2009; Kohlstedt et al., 2009; Sarmiento-Gomez et al., 2012). These properties are different for Pf1 phage, wild-type fd, and modified fd, and it is interesting to relate these differences in liquid-crystal properties to differences in the virion structure. For instance, the difference in liquid-crystal properties between wildtype fd and the Y21M mutant (Barry et al., 2009) may be related to the difference between the fdD and fdC symmetry of the individual virions, discussed in Section 2.1. The structure, assembly and structural transitions of the filamentous phage virion involve a widespread motif in structural biology, the a-helix bundle, and the potential value of filamentous phage as a model has long been recognized. More detailed structural analysis is often possible for filamentous phage than for many other fibrous proteins, and may lead to reinterpretation of structures involving arrays of a-helices. We close by repeating the remark made in an earlier review: “If we provoke some workers sufficiently to cause them to do experiments to disprove our models, the review will have had some value” (Marvin and Hohn, 1969). Acknowledgements We would like to acknowledge F.J. Marvin’s suggestions about early drafts. SKS acknowledges funding from the Natural Sciences and Engineering Research Council of Canada and a Marie Curie International Incoming Fellowship. Appendix A. Supplementary material Supplementary material related to this article can be found at http://dx.doi.org/10.1016/j.pbiomolbio.2014.02.003. References Ackermann, H.-W., 2006. Classification of bacteriophages. In: Calendar, R. (Ed.), The Bacteriophages, second ed. Oxford University Press, New York, pp. 8e16. Agris, P.F., Vendeix, F.A.P., Graham, W.D., 2007. tRNA’s wobble decoding of the genome: 40 years of modification. J. Mol. Biol. 366, 1e13. Aisenbrey, C., Harzer, U., Bauer-Manz, G., Bär, G., Chotimah, I.N.H., Bertani, P., Sizun, C., Kuhn, A., Bechinger, B., 2006. Proton-decoupled 15N and 31P solid-state NMR investigations of the Pf3 coat protein in oriented phospholipid bilayers. FEBS J. 273, 817e828. Alberts, B., Frey, L., Delius, H., 1972. Isolation and characterization of gene 5 protein of filamentous bacterial viruses. J. Mol. Biol. 68, 139e152.


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Structure and assembly of filamentous bacterial viruses.

Controlling the Morphology of Organic Crystals with Filamentous Bacteriophages.

Filamentous Bacteriophage Promote Biofilm Assembly and Function.

Structural polymorphism correlated to surface charge in filamentous bacteriophages.

Genomic characterization and integrative properties of phiSMA6 and phiSMA7, two novel filamentous bacteriophages of Stenotrophomonas maltophilia.

Flow linear dichroism spectra of four filamentous bacteriophages: DNA and coat protein contributions.

2.

Secondary structure of RNA from bacteriophages f2 Qbeta, and PP7.

Ubiquitination and filamentous structure of cytidine triphosphate synthase.

The structure and assembly of microtubules.

Aspects of gap junction structure and assembly.

Structure and assembly of phage phi29.

Structure, morphology, and assembly behavior of kafirin.

Assembly and structure of the T3SS.

Packaging of genomes in bacteriophages: a comparison of ssRNA bacteriophages and dsDNA bacteriophages.

Photoreactivation of bacteriophages after UV disinfection: role of genome structure and impacts of UV source.

Oligomeric assembly is required for chaperone activity of the filamentous γ-prefoldin.

Draft Genome Assembly of Filamentous Brackish Cyanobacterium Limnoraphis robusta Strain CS-951.

Neutron scattering study of the solution structure of bacteriophages Pf1 and fd.

Structure and assembly of the capsid of bacteriophage P22.

Surface appendages of archaea: structure, function, genetics and assembly.

Assembly and structure of Lys33-linked polyubiquitin reveals distinct conformations.

Structure and assembly of scalable porous protein cages.

Structure, Function, and Assembly of Adhesive Organelles by Uropathogenic Bacteria.