Biochimie ( 199 i ) 73, 227-230

© Soci6t6 franqaise de biochimie et biologie mol6culaire / Elsevier, Paris

227

The solution structure of recA filaments by small angle neutron scattering P A T i m m i n s l, R W H R u i g r o k 2, E D i C a p u a 2 llnstt;ut Laue Langevin, 156X. 38042 Grenoble Cedex; 2EMBL, Grenoble Outstatirm. c/o ILL. 156X, 38042 Grenoble Cedex. Frunce

(Received 25 January 1991, accepted 1 February i 991 )

Summary - - The technique of small angle neutron scattering has been applied to study the structure in solution of recA self-polymers

and various recA-DNA complexes. These results are compared with those recently obtained by other physical techniques. RecA / neutron scattering / filaments / recA-DNA complexes

Introduction

Purified recA protein of E coli (MW 38 kDa) has 3 DNA-dependent activities in vitro: hydrolysis of ATP, cleavage of LexA repressor protein in the presence of ATP (or the non-hydrolyzable analogue ATI>?S) and strand exchange between the resident DNA and an incoming DNA (also dependent on ATP (ATPyS); for a review see [!]). In order to elucidate the mechanism of these reactions, we have studied the structure of recA under reaction conditions. We review here the recent results obtained using neutron small angle scattering to study the structure in solution of filamentous complexes of recA with DNA and of self-polymers. The results are compared with the results obtained by electron microscopy and in the crystal. As neutron scattering is a technique that is rather poorly known to biologists, we have taken this opportunity to present in outline the fundamental aspects of the method. Neutron scattering

Neutron small angle scattering is a technique which may be used to investigate the size and shape of biological particles in aqueous solution in a non-perturbative manner (due to the low energy of thermal neutrons there is no radiation damage incurred by the sample; for a review of applications see [2]). In its basic theory, neutron small angle scattering is analagous to X-ray small angle scattering as described by Guinier and Fournet [3]. Thus, a solution of macro-

molecules when irradiated by a neutron or X-ray beam will scatter the incident radiation at small angles from the main beam (< - 1 0 °) in a way that depends on the size and shape of the panicles. The difference between X-rays and neutrons lies in the mechanism by which the different radiation is scattered by different atoms or groups of atoms (and in the wavelength of the incident radiation). The scattering power of atoms for X-rays depends on the atomic number of the atom concerned (since it is the electrons that act as scattering points), whereas for neutrons the scattering power depends on the structure of the atomic nucleus. Thus, for example, the scattering power of hydrogen and deuterium are identical for X-rays but extremely different for neutrons. As all biological mo!ecules, as well as their aqueous environment, contain large numbers of hydrogen nuclei, this difference in scattering power can be exploited to change the scattering of groups of atoms whenever hydrogen can be replaced by deuterium. In all radiation scattering techniques (X-ray, EM, optical, neutrons) an object is visualized through the contrast between itself and its environment. In the case of neutron scattering, this contrast comes froin the difference in scattering power between the macromolecule and its aqueous environment. As mentioned above, the scattering density of the macromolecule or the aqueous environment can be changed by manipulating the hydrogen/deuterium content of the water or the macromolecule or both. We may consider the change in the scattering density of the water as analogous to negative staining in EM and a deuteration of the macromolecule as being similar to positive staining. The macromolecule is,

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ho~,.ever, not perturbed as is the case for EM, because deuteration is performed by the bacterium during grovcth in D_,O medium. In our stadies of recA both these approaches of enhancing contrast approaches have been used. In vivo deuterated DNA has been used to locate the DNA within recA/dsDNA complexes [41 and a D,O medium was used to enhance the contrast when studying the helical parameters of a wide range of different filaments of recA with and without DNA [5]. A further characteristic of this contrast effect is that it allows studies to be made in solutions of high salt concentrations, where X-ray and EM studies would be impracticable [61. The classical theory for the analysis of small angle scattering in solution was developed for the case of a monodisperse system of identical particles in solution. Two quantities can be measured in such an experiment which are completely independent of any preconceived model: the radius of gyration and the molecular weight. (The radius of gyration is the massweighted radius: R?, = E mi ri2/E mi) A similar analysis is also possible for rod-like particles [71. Here the theory holds as long as all rods have the same cross-sectional structure and are more than = 5 times as long as they are wide. In this case the quantities derived are the cross-sectional radius of gyration Rc and the mass per unit length. The crosssectional radius of gyration is a measure of the radial distribution of mass around the axis of the rod. In the simple case of a homogeneous rod, R~ = ~---x R where R is the rod radius. For the case of helical particles, the relationship is much less straightforward and depends on details of the structure; the R c is however d;agnostic for a particular form of filament, in addition, in the case of helically symmetrical particles, the theory for small angle scattering by solid rods must be applied with caution as there may be contributions from the helically repeating elements in the structure. The interpretation of the scattering curves in terms of the cross-sectional radius of gyration is only applicable within a limited angular range, where Q x R~ < 1 (Q = 4n sin 0/Z: 20 = scattering angle, k = incident neutron wavelength). The scattering curve is however measurable at much larger angles (Q-values) and this wider angle data contains further structural information. In this range the method of interpretation is to find a model whose calculated scattering curve best corresponds to the measured data. This approach, although it cannot provide a unique solution, can select the most realistic models, particularly when used in conjunction with other techniques. In the case of recA, we fitted the observed scattering curves by models in which the recA subunit was approximated by a 22.5 A radius sphere and this sphere repeated by helical symmetry. This model can be described by only 3 parameters, the number of units per turn, the

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radial location of the subunit and the helical pitch. Figure 1 shows a typical experimental scattering curve of scattered intensity vel'slts scattering angle; it is obtained by circular averageing of the data collected on a planar detector. The regions are indicated which may be used for calculating crosssectional radius of gyration (region a) and for modelfitting (region b). Note the prominent maximum at high Q which is due mainly to the helical repeat of the structure.

Results For different complexes of recA, the cross-sectional radii of gyration and the mass per unit length (M/L, calculated from the Guinier zone) as well as the helical pitch derived from the high angle maximum (by model fitting) are shown in table I. We see from this data that the filaments fall essentially into 2 categories which we describe as

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Fig 1. Scattering curve from recA self-polymers (compact form), obtained from the superimposition of data collected at very small angles (the 'Guinier range', (a), from which M/L and Re are calculated) and wider angles (subsidiary maximum). The 2 regions are shown and discussed separately in [5]. The whole of region (b) is used fol' model fittillg.

RecA filaments solution structure

Table I. Parameters obtained by small angle neutron scattering on filaments of recA self-polymers and complexes with DNA (from DiCapua et al [4-6]. The samples were all measured at a concentration in the order of 4 mg/ml recA, in a buffer (20 mM potassium phosphate pH 6.8) containing about 5% glycerol, 2 mM Mg(acetate)2 and, where appropriate, 1 mM ATP'~S and DNA in slight excess (4.5 bp dsDNA/recA, 6-9 nt ssDNA/recA: for discussion see [5]).

R, (A)

Pitch M/L (A) recA/lO0

recA self polymer

+ATPyS -ATPyS +ATPyS +salt

39 39 34

70-75 70-75 95

6.4 6.2 nd

recA ss DNA

+ATPyS -ATPyS

33 39

95 70--75

5.8 6.5

recA ds DNA

+ATPyS -ATPyS

33 95 No complexes

5.5

'compact' (Rc = 39 A) or 'open' (Re = 33 A). The higher angle scattering curve can best be described for the compact state by a helix of 70-75 A pitch with subunits placed at a radius of 35 A,. The open structure is described by a helix of 95 A pitch with subunits at 28 A, radius. For computational reasons, the helices both contain integral numbers of subunits per turn, 5 for the compact and 6 for the open form. The real structure could of course deviate somewhat from this. Indeed, the M/L in table I do not fit these numbers perfectly; as the calculation of M/L depends directly on the value of the concentration of macromolecule in the sample, it is liable to error [5]. What appears to be reproducible, however, is that the compact and open structures of the recA filaments differ by a factor of about 1.1-1.2 in their M/L.

Conclusions and comparison with other structural results Our small-angle scattering data support the idea that recA filaments exist in 2 major conformational font, s: the open and the compact filament, 'open' with 95 A pitch being characteristic for the enzymatically active complex of recA with D N A (both ss and ds) and ATP, and 'compact' with 70-75 A pitch being characteristic for inactive forms of recA. The existence of 2 forms has recently also been discussed by Heuser and Griffith [8] and Lee and Cox [9]. Within the two classes of conformation, our data allow for = 10% fluctuation for all 3 parameters (Re + 1.5 A, M/L + 0.5 units, pitch + 5 A). However, we do

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not find intermediate forms and therefore do not think that the 2 conformations change into one another without a major rearrangement, involving eg all subunits at the same time. It is of interest however to note that in the crystal ([10]; Story and Steitz, personal communication), the recA units are arranged in a continuous helix of 6.0 recA per turn of 83 A pitch, ie, halfway between our compact and ~,pen solution structures. Could this be due to crystallization constraints (packing forces and the pH of crystallization, pH 5) acting on the 5.5 recA per turn of 75 A pitch of the compact form? Indeed, 5 or 5.5 recA per turn could not pack into any normal crystal symmetry. On the other hand, Yu and Egelman [11] have suggested the helix form in the crystal to be the limit case of the flexibility of the open helix of 6.17 recA per turn of 95 A pitch, having observed by EM-reconstructions of negatively stained complexes with DNA a variation between segments in the range of 6.1-6.2 recA per turn of pitch 88-93 A. We favour the argument of the crystallization constraints since the crystal was produced in the absence of DNA and ATP and is therefore expected t,3 be more cognate to the inactive form (our compact structure). In another respect also, our solution structure of the compact form needs discussion: by electron microscope', the pitch of this form is found to be 55-75 A, (64 A, [121; 55 or 75 A,, [13]; 60 A, [8]). In [5], we have shown evidence that this small pitch might be an EM artefact, due either to chemical crosslinking or to adsorption to the carbon film. Indeed, just as crystallization may force the 5.5 recA per turn into 6, it is llLIL

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ture (the compact form is compact only by outer appearance since it is expected to contain 60-70% water as estimated from its mass and diameter) may shrink the pitch, and perhaps adsorption may force the 5.5 recA per turn into 5. Confirmation comes from EM samples in amorphous ice, where both fixation and adsorption are avoided: with our samples, ie our buffer conditions, E Hewat (personal communication) has recently observed a pitch of 75 + 1 A for both the self-polymer and the complex with ssDNA in the absence of nucleotide cofactor (confirming the finding by Chang et al [14], on the complex with ssDNA in the absence of ATP). The more interesting structure of course is that of the active complex with 6.i7 recA per turn of 95 'A pitch; neutron scattering has contributed in this context the structural analysis of the self-polymer in high salt (which is active in ATP hydrolysis [151 and, in the present, c of ATPyS, LexA repressor cleavage [6]), thanks to the unique property of neutrons to 'see" contrast under conditions less accessible to X-rays or conventional EM. Finally, the bona fide active form

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of rccA. the c o m p l e x with D N A in the presence of A T P (ATP),S), has been found to have essentially the same low resolution structure by conventional E M , by solution scattering, and by E M in a m o r p h o u s ice.

References ! 2 3 4

5

6 7

Roca AI, Cox MM (1990) The recA protein: structure and function. CRC Critical Rev (in press) Timmins PA. Zaccai G (1988) Low resolution structures of biological complexes studied by neutron scattering. FmBiophys J 15, 257-268. Guinier A, Fournet G (1955) Small Angle Scattering o f Xrays. Wiley. NY D~Capua E, Schnarr M, Timmins PA (1989) The location of DNA in complexes of recA protein wi~h double stranded DNA. A neutron scattering study. Biochemistry _8, 3287-3292 DiCapua E, Schnarr M, Ruigrok RWH. Lindner P,Timrains PA (1990) Complexes of recA protein in solution. A study by small-angle neutron scattering. J Mol Biol 214, 557-57() DiCapua E, Ruigrok R.H, Timmins PA (1990)Activation of recA protein: the salt induced structural transition. J Struct Biol 104, 91-96 Torbet J, Gray DM, Gray CW, Marvin DA, Siegrist H (1981 ) Structure of the fd DNA-gene 5 protein complex in

solution. A neutron small angle scattering study. ,I Mol Biol 146, 305-320 8 Heuser J, Griffith J (1989) Visualization of recA protein and its complexes with DNA by quick freeze/deep etch electron microscopy. J Mol Biol 210, 473-484 9 Lee JW, Cox MM (1990) Inhibition of RecA protein promoted ATP hydrolysis. !. ATPyS and ADP are antagonistic inhibitors. Biochemistry 29, 7666-7676 10 McKay DB, Steitz TA, Weber IT, West SC. HowardFlanders P (1980) Crystallization of monomeric recA protein. J Biol Chem 255, 6662 11 Yu X, Egelman EH (1990) Image analysis reveals that Escherichia Coli recA protein consists of two domains. Biophys J 57,555-566 12 Stasiak A, Egelman EH (1986) Structure and dynamics of recA protein-DNA complexes as determined by image analysis of electron micrographs. Biophys ,I 49, 5-6 13 Williams RC, Spengler SJ (1986) Fibres of recA protein and complexes of recA protein and single stranded ~X 174 DNA as visualized by negative stain electron microscopy. J MolBiol 187, 109-118 14 Chang CF, Rankert DA, Jeng TW, Morgan DG, Schmid MF, Chiu W (1988) Cryo electron microscopy of unstained unfixed recA---css DNA complexes. J Ultrastruct Mol Struct Res 100, 166-172 15 Pugh BF, Cox MM (1988) High salt activation of recA protein ATPase in the absence of DNA. J Biol Chem 263(1 ), 76-83