Brave new proteins: what evolution reveals about protein structure Annalisa Pastore and Arthur M. Lesk EMBL, Heidelberg, Germany and University of Cambridge, Cambridge, UK Synthetic proteins provide important information about the principles of protein structure. They illuminate the processes of natural protein evaluation, but are not limited by these processes. Here, we review studies of several mutant proteins and discuss the general principles that can be derived from them. Current Opinion in Biotechnology 1991, 2:592-598 Introduction Our understanding of protein architecture is based primarily on the analysis of structures of natural proteins, as determined by X-ray crystallography or nuclear magnetic resonance. Recent syntheses of novel proteins by genetic engineering have extended our appreciation of the amino acid sequences compatible with particular folding pattems. Analyses and syntheses are carried out to explore the relationship between amino acid sequence and protein conformation. Synthetic work can be used to explore the set of sequences compatible with a folding pattern more completely, however. This is partly because the set of natural known sequences is incomplete, but also because constraints on pathways of natural evolution may render inaccessible some sequences that are potentially compatible with a folding pattern. Synthetic work is not subject to these constraints. Why can molecular evolution in nature explore only a limited subset of the amino acid sequences that would potentially fold to form homologous proteins? Even in protein families such as the globins, cytochromes c or serine proteases, for which we have a fair sampling of homologous molecular structures from distantly related species, two factors limit the observed structural variation: physical laws that govern the stability of protein structures (obviously these apply equally to synthetic proteins), and constraints on evolutionary pathways. For any two natural related protein structures, pathways must exist by which they have evolved from a common ancestor, through molecules with amino acid sequences differing by one or at most a very few sequence changes, that have all been subject to the test of natural selection that they be functional. (We exclude large-scale block transfers of entire regions'or domains.) The ability to construct non-natural proteins by genetic engineering has thus widened the universe of accessible sequences. Functional proteins produced synthetically are not limited by constraints on evolutionary pathways, or by any other features of natural proteins that
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are accidents of history. This situation has implications for biotechnology. In order to decide which structural features might be reasonable design targets for synthetic proteins, it is important to try to understand the constraints imposed by natural evolution and to recognize historical accidents (to avoid being "condemned to repeat them"). Conversely, the synthesis and assessment of the structural and functional properties of non-natural proteins can help to distinguish physical law from historical accident.
Natural molecular evolution Studies of natural protein evolution have shown that, as amino acid sequences diverge by mutations, insertions and deletions, the protein conformation changes [1]. The larger the divergence of the sequences, the larger the distortion of the conformations. Several observations have been made about the divergence of homologous proteins during evolution [2,3]. First, a core of the secondary structure, including the active site, retains its folding pattern. Other regions of the molecule may refold entirely. Second, although individual helices or sheets within the core tend to retain standard conformations, they shift and rotate with, respect to one another. This happens because stability of the protein requires formation of well packed interfaces between elements of secondary structure. When mutations alter residues at interfaces, the packing must adjust. Typically, there is room to accommodate the extra odd methyl group without significantly changing the main-chain conformation. But (for example) a substantial increase in side-chain volume at an interface will tend to push the helices or sheets apart. A third observation is that the pattern of residue-residue contacts at interfaces tends to be preserved. This is consistent with evolutionary pathways in which substitution of one residue at a time allows the retention of a well packed interface by adjustment of the relative geometry of the interacting helices or sheets. The pattern of
(~ Current Biology Ltd ISSN 0958-1669
Brave new proteins: what evolution reveals about protein structure Pastore and Lesk S93 residue-residue interactions at an interface influences the relative geometry of the elements of seconda~ structure. For packed helices, for example, the structural pattern at the interface is correlated with a range of interaxial angles. This suggests that the conservation of the pattern of residue interactions is not merely evolutionary stodginess but may be essential either for the stability of the native state or possibly for the folding pathway. Fig. 1 summarizes schematically the progressive divergence of sequences within a family of proteins. Amino acid sequence similarity, measured by the number of identical residues in the correct sequence alignment of a pair of proteins, decreases from 100% on the left to below the noise level on the right. Down to about 40-50% sequence identity the structures are very similar. From 20-40% sequence identity, the basic fold of the core of a protein family has a conserved topology but the structures are distorted. Below 20%, one cannot be sure from sequences alone that two proteins are homologous: the fold may well be conserved although the homology cannot be recognized in the sequences. That is, only proteins with above 20% sequence identity can be identified as homologous, but proteins with below 20% sequence identity cannot be assumed not to be homologous.
Proteins definitely homologo Structures very similar _< 1.0 ~ root mean square main-chain deviation
General
similarity of fold
I
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0 % Identical amino acids
Fig. 1. The relationship between divergence of amino acid sequence and divergence of structure in proteins. For simplicity, we consider three classes of closeness of relationship (the boundaries are only approximate): from 100% residue identity down to 50%, protein structures are very similar; from 50% to 25% residue identity, the proteins are definitely homologous but their structures show substantial differences; below 25%, proteins may be homologous and similar in structure or may be unrelated.
Very distantly related proteins often arise through evolution with change of function. During evolutionary events that procede with retention of function, global constraints on the structure of a protein link the geometric changes at interfaces between elements of secondary structure [ 1]. In evolution with change in function, these constraints are released, or, rather, replaced by other constraints. Two sequences can diverge to the point at which the relationship between them cannot be recognized, and the structural changes are much larger than in closely related proteins. The crystallins provide a good example of this process at work. A variety of enzymes have been recruited for structural roles in lenses of ver-
tebrates; some but not all of them retain their enzymatic av ....
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[qj.
Synthetic proteins Synthetic single-site mutations can be carried out to explore novel sequences in a way comparable to evolution. (For reviews see [5,6", 7]). In contrast, multiple simultaneous amino acid substitutions allow the exploration of sequences inaccessible to natural evolution. There have been a number of studies of multiple synthetic mutants, notably by Sauer and coworkers [8,9,10",11"-13"]. On the computational side, Lee and Levitt [14,] have been able to predict the relative stabilities of some of these mutants. Ponder and Richards [15] have written programs to search for alternative packings of a hydrophobic core, and applications of this program to synthetic work have begun.
Structural studies of synthetically mutated proteins X-ray crystallography makes it possible to describe in detail the structural consequences of mutations and to correlate them with changes in stability and function. The combination of mutagenesis and structure determination makes it possible to probe selected structural features systematically. Areas of recent progress include (but are not limited to): the quality of hydrophobic packing in protein interiors and its effect on stability; addition or deletion of disulfide bridges; and changes in function associated with mutations at active sites. Matthews and coworkers [16-20] have made mutants of T4 lysozyme and solved many of them to high resolution. To explore the effect of quality of packing in the interior, they increased the size of a side chain adjacent to either of the two largest cavities within the structure. The two mutants, lmu133Phe and Ala139Val, had normal activity but slightly reduced stability (melting temperature lowered by 1-3°C). The structural perturbations were small (largest shift about 0.7g,) and localized near the site of mutation; in the Ala139Val mutant a portion of an a-helix shifts concertedly by 0.15g~ In each case, the mutant side chain is somewhat strained, accepting non-ideal sidechain torsional angles and mildly uncomfortable steric interactions, thus offsetting the gain in stability arising from burying additional non-polar atoms. It is interesting that the structure is only partially able to 'relax' around the mutations. In related work, the structures of two T4 lysozyme mutants Ile3Val and Ile3Tyr were examined [ 17]. In the wildtype, Ile3 sits in a hydrophobic pocket; only its C2-2 atom is solvent-accessible. Reducing the side chain volume in Ile3Val produced a structure 'essentially identical to the wild-type'. In contrast, the tyrosine side chain at this site
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Proteinengineering does not fit into the pocket, and turns out into the solvent, a water molecule entering the cavity left behind. Conformational changes include shifts in side chains adjacent to the site of mutation and concerted shifts in two helices, by about 0.6-1.0A. Structural studies of the consequences of additions and deletions of disulfide bridges have been carried out in T4 lysozyme [18-20], bovine pancreatic trypsin inhibitor [21] and subtilisin BPN' [22]. Disulfide bridges inserted into T4 lysozyme at residue pairs 9--164, 21-142 and 3-97 increase stability, additively; the triple disulfide mutant has a melting temperature 23.4°C higher than the wildtype. Removal of disulfide bridge 30-51 in bovine pancreatic trypsin inhibitor by changing both cysteines to alanines showed localized shifts in two segments of chain (about five residues surrounding each site of mutation) to try to fill the gap [21]. The structural analysis of functional changes arising from mutations of active-site residues is seen in mutants of a-lytic protease [23] and tyrosyl-tRNA synthetase [24]. Alpha-lytic protease is a serine protease with a specificity for alanine in the P1 site, which is produced by occupancy of the specificity pocket by the side chain of Met192. The mutants Metl9ZAla and Met213Ala (Met213 is adjacent to the specificity pocket) showed broadened specificity, arising from removal of the large side chain and small changes in and around the binding pocket. Confonnational changes were observed, both with and without ligand, an-d it was pointed out that the ability of the structure to deform to accommodate ligands enhanced the range of specificity. What can we learn from these structures? We know that proteins are somewhat plastic, from comparisons of structures of proteins in different crystal forms, which show the deformations induced by different crystal packing forces [2,3]. Indeed, Eigenbrot, Randall and Kossiakoff [21] point out that the structural changes observed in mutant proteins "are of the same order of magnitude or smaller than those associated with the lattice forces alone". In general, in response to a mutation, the structure tries to deform to compensate. Of course, this cannot always be carried out successfully. The repertoire of available responses includes local deformation and binding of solvent in cavities created. Several authors [18-20,22] have noted concerted shifts in a-helices, which have also been observed in response to crystal packing forces and in conformational changes arising from changes in ligation states [25-27]. Thus, the capacity of proteins for structural change is important not only for permitting evolution to explore sequence changes, but also for functional applications, for example in enzymes that show 'induced fit' or allosteric changes (recently reviewed by Perutz [28-,]).
Multiple mutagenesis as a probe of sequences compatible with a fold Lim and Sauer [9,10"] have used cassette mutagenesis to randomize selected sets of seven residues in the hydrophobic core of the amino-terminal domain of phage £-repressor limiting their substitutions to the large hydrophobic residues valine, leucine, isoleucine, methionine and phenylalanine. Selecting for functional molecules followed by sequencing produced a catalogue of sequences that are compatible with the native fold and showed that most form reasonably stable structures although, in many cases, with reduced activity. Several properties of the core residues essential for folding were noted, including retention of hydrophobicity. A restraint in total volume changes was also observed: the total volume of the core residues was found to lie within the equivalent of two extra or three fewer methylene groups of the wild-type. Finally, a 'steric constraint' was found to exist, in that different sequences with similar core hydrophobicity and total side-chain volume were not always functionally equivalent. That is, the requirements for function are more stringent than the requirements for the general overall folding pattern. For example, three residues in the core contain, in the original protein, Va136, Met40 and Val47. The single mutant Met40Leu is functional but the double mutant of the same amino acid composition Va136Leu/Met40Val is only partially functional. The authors interpret these differences in terms of the steric quality of the packing of these residues. Clearly the structural shifts produced by the different packings are affecting the relative spatial disposition of groups in these proteins important for function. Other studies by this group based on the same technique include the exploration of acceptable substitutions in a helix interface in the ~v-repressor [12.], and in a molecule containing a leucine zipper, constructed by fusing the GCN4 leucine zipper domain with the £-repressor. The role of the repressor in these experiments is primarily to assay for dimerization of the leucine zipper domain [13"].
Structural implications of packing constraints Lacking the structures of the synthetic multiple mutants of the ~,-repressor, we illustrate steric constraints on packing in natural proteins. Chothia (see [29]) showed that interfaces between packed helices in protein interiors are not merely 'hydrophobic glue' but have specific structures. The side chains on the surfaces of the helices form well defined ridges and grooves, and interfaces form by packing ridges on the surface of one helix into grooves of the surface of the other. There are three main ways of forming ridges on a helix surface, and their interactions give rise to distinct classes of interface patterns, with typical interaxial angles.
Brave new proteins: what evolution reveals about protein structure Pastore and Lesk 595
(b)
.
,
Fig. 2. Van der Waals slices showing (a) the packing at the interface between B and G helices in sperm whale myoglobin, showing the most common type of 'ridges-into-grooves' packing and (b) the packing at the interface between B and E helices in sperm whale myoglobin, showing the unusual 'crossedridge' structure of the interface, in which ridges from the two helices cross over a 'notch'. Residues from the B helix are shown as solid lines; those from the G helix (a) and E helix (b) are shown as broken lines. Thick lines indicate ridges. Two examples illustrate the structural specificity of the packing [30]. Fig. 2(a) shows the interface between the B and G helices of sperm whale myoglobin, illustrating the most common type of 'ridges-into-grooves' packing. In contrast, Fig. 2(b) shows the interface between the B and E helices of myoglobin, which has an unusual 'crossed-ridge' structure, a packing pattern in which ridges from the two helices cross over a 'notch'. The ridges are formed by the residues Va121, Gly25, Leu29 and Phe33 (from the B helix) and Leu61, Gly65 and Leu69 (from the E helix). Each glycine residue forms a notcl'f in its ridge. The two glycine residues sit over each other, allowing the ridges to cross without loss of good packing. This structure imposes a constraint on the amino acid sequences, in terms of the relative volumes of adjacent residues on the helix surface. Studies of homologous proteins show that as protein families diverge, the class of the packing is retained. This is shown in Fig. 3, for the helix contacts in the globins. This conservation also extends to phycocyanin [31o.].
Role of packing in determining structure Behe et al. [32] have argued recently that packing does not determine the native fold. Although there is a consensus that stabilization of the native state requires efficient packing of hydrophobic residues in the protein interior, the authors suggest that these interactions have little specificity, and that many combinations of hydrophobic residues could form well packed interiors of comparable stability. Given the extreme form of this premise that quality of packing is sequence independent, then provided buried residues remain hydrophobic, it would follow that the three-dimensional structure cannot be determined by the identities of the residues at the buried sites;
Behe et al. [32] have presented statistical data on the interactions of residue pairs in natural proteins of known structure. They were looking for two types of relationships, the first being preferred pairwise complementarity of side chains, that is, residues that, because of mutual conformability, are preferentially in contact within protein interiors (e.g."... leucine residues might nestle together like spoons ..." [32]), and the second being preferred pairs of residues that pack unusually efficiently with their surroundings. No such preferences, in either case, were found. In the context of this review, it should be noted that Behe et al. are in fact discussing native proteins, and not the potentially more general sets of sequences that might produce the same fold but are inaccessible to natural evolution. Their statistical analysis of residue interactions is based on crystal structures of natural protein structures. Their suggestion that it is patterns of hydrophobicity that define conformation is based on the feasibility of classifying natural sequences on the basis of hydrophobicity pattems [33,34"]. Behe et al. conclude that specificity in residue packing cannot determine protein conformation. In other words, packing interactions do not constrain the sequences that will produce a given folding pattern. In natural proteins, however, there are structural constraints on protein interiors (Fig. 2b), in which the relative volumes of residues create the notch that permits the conserved crossed-ridge structure. Comparisons of related structures show that the conservation patterns are considerably more subtle than pairwise correlations of residues (except for disulfide bridges) [35]. Analyses of aligned sequences for patterns of hydrophobicity that define folding pattems do, in fact, include restraints or even constraints on residue volumes at certain positions [33,34"]. For example, Bowie et al. [34"] show that patterns derived from aligned sequences form identifying 'signatures' of folding pattems for globins, cy-
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tochromes c, CheY and calcium-binding proteins. Their patterns represent residues by a three-letter 'alphabet' corresponding to classes of hydrophobicity according to the scale of Fauchere and Pliska [36]. The three classes of residues are WIPI_MVC, YPATHGS and QNEDKR (residues identified by one-letter codes). In fact, these classes do segregate residues by volume as well as by charge or polarity. The structural constraints observed in natural molecules do not preclude the possibility of other modes of packing consistent with a given fold, which might accommodate a wider range of sequences. These might arise in ge netic engineering experiments such as those of Sauer and coworkers [8,9,10oo,11.-13°], or be suggested by calculations of the type of Ponder and Richards [15]. The specificity in packing interactions that are observed in known structures may be partly historical accidents, and other structures, showing less specificity, may well be stable. An example is discussed in the next section. Until such possibilities are more fully explored, however, it would seem premature to exclude packing constraints from any possible role in determining folding patterns.
Non-natural globins: a challenge It is possible to detect, in Fig. 3, an opportunity to create a structure that might be functional, but not accessible to evolution from known natural globins. Although
Fig. 3. The distribution of interaxial distances and angles in the major helix contacts in the globins. Homologous pairs of helices retain the structure of the interface, in terms of the pattern of residue packing. Letters signify pairs of packed helices - - for example, B-G indicates the interaction between the B and G helices. Symbols at large dots indicate the species of origin of the globin. Ha, human haemoglobin a-chain; H~, human haemoglobin 1[3chain; Ea, horse haemoglobin a-chain; E~, horse haemoglobin [[]-chain; W, sperm whale myoglobin; L, sea lamprey globin; G, Glycera globin; C, Chironomus erythrocruorin; Lg, lupin leghaemoglobin [15].
homologous helix interfaces cluster in interaxial distance and angle, the B-E and B-G classes, which have different modes of residue packing, approach each other. For example, the B-E interface of Glycera globin has an interaxial distance and angle very similar to that of the B-G contact in Cbironomus erythrocruorin, although the pattern of residue interactions is different. This suggests that it would be interesting to try to transfer the interface; that is, to create a non-natural globin by modifying (for example) Glycera globin with multiple simultaneous mutations so that the B-E interface contains the residues and the packing of the B--G interface of Chironomus erythrocruorin, or perhaps some other such pair that appears to be the most compatible structurally. (This is an example of the kind of alternative packings that the methods of Ponder and Richards [15] are designed to detect.) If this experiment were successful it would produce a globin that, as far as we know, is inaccessible to natural evolution.
Conclusion Natural protein evolution imposes constraints on protein structures that do not apply to molecules created by genetic engineering. An understanding of such constraints can suggest what kinds of structures might be achievable synthetically. The creation of non-natural structures is necessary to illuminate the principles that underly the features of natural ones.
Brave new proteins: what evolution reveals about protein structure Pastore and kesk
Acknowledgements We both thank C Chothia and AG Murzin for helpful comments. AM Lesk also thanks the Kay Kendall Foundation for generous support.
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest •• of outstanding interest CHOTHIA C, LESK AM: The Evolution of Protein Structures. Cold Spring Harbor Symp Q u a n t Biol 1987, LII:399-405. LESKAM, CHOTHIAC: The Response of Protein structures to Amino Acid Sequence Changes. Phil Trans Roy Soc London 1986, 317:345-356. 3.
CHOTH1A C, LESKAM: Relationship B e t w e e n t h e Divergence of Sequence and Structure in Proteins. ~ B O J 1986, 5:823-826.
By random mutagenesis the sequence requirments of 25 positions in the amino-terminal domain of ~.-repressor are investigated. A variety of degrees of tolerance was observed. O n the suface, s o m e positions are very robust and will accept essentially all types of residues. Others accept only hydrophilic residues and one requires proline. Buried residues are constrained relatively stringently. 13. •
Hu JC, O'SHEA EK, KIM PS, SAUERRT: Sequence Requirements for Coiled-Coils: Analysis w i t h ~ Repressor--GCN4 Leucine Zipper Fusions. Science 1990, 250:1400-1403. Eight positions in the hydrophobic interface of the leucine zipper dimer from GCN4 were subjected to mutation, in order to determine the acceptable range of variability at these sites. Most single mutants that retained hydrophic character, but not all multiple mutants were functional. Leucine was not essential at the special positions but was favoured. LEE C, LEVITt M: Accurate Predication of t h e Stability and Activity Effects of Site-Directed Mutagenesis o n a Protein Core. Nature 1991, 352:448-450. Stabilization energies of 78 triple mutants of X-repressor have been calculated and are well correlated with the thermostabilities of the mutants. Active and inactive mutants are discriminated with 92% accuracy. 14. .
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PONDER JW, RICHARDS FM: Tertiary Templates for Proteins: Use of Packing Criteria in t h e Enumeration of Allowed Sequences for Different Structural Classes. J Mol Biol 1987, 193:775-791.
PIATIGORSKY J, WISTOW G: T h e R e c r u i t m e n t of Crystallins: New Functions Precede Gene Duplication. Science 1991, 252:1078~1079,
16.
KARPUSAS M, BAASE WA, MATSUMURA M, MATrHEWS BW: Hydrophobic Packing in T4 Lysozyme Probed by CavityFilling Mutants. Proc Natl Acad Sci USA 1989, 86:8237-8241.
ALBER T: Mutational Effects o n Protein Stability. A n n u Rev Biochem 1989, 58:765-798.
17.
MATSUMURAM, WOZNIAKJA, SUN DP, MAT17-IEWSBW: Structural Studies of Mutants of T4 Lysozyme that Alter Hydrophohic Stabilization. J Biol Chem 1989, 264:16056-16059.
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MATSUMURAM, SIGNOR G, MATTHEWSBW: Substantial Increase of Protein Stability by Multiple Disulphide Bonds. Nature 1989, 342:291-293.
19.
MATSUMURAM, BECKTEL WJ, LEVITT M, MATnmWS BW: Stabilization of Phage T4 Lysozyme by Engineered Disulfide Bonds. Proc Natl Acad Sci USA 1989, 86:6562q5566.
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PJURA PE, MATSUMURAM, WOZNIAKJA, MATrHEWS BW: Structure of a Thermostable Disulfide-Bridge Mutant of Phage T4 Lysozyme Shows that an Engineered Cross-Link in a Flexible Region does not Increase t h e Rigidity of the Folded Protein. Biochemistry 1990, 29:2592-2598.
21.
EIGENBROT C, RANDALLM, KOSSIAKOFFA& Structural Effects I n d u c e d by Removal of a Disulfide-Bridge: t h e X-Ray Structure of the C30A/C51A Mutant of Basic Pancreatic Trypsin Inhibitor at 1.6A. Protein Eng 1990, 3:591-598.
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KATZ B, KOSSIAKOFFAA: Crystal Structures of Subtilisin BPN' Variants Containing Disulfide Bonds and Cavities: Concerted Structural Rearrangements I n d u c e d by Mutagenesis. Proteins 1990, 7:343>357.
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BONE R, S1LENJL, AGARD D& Structural Plasticity Broadens t h e Specificity of an Engineered Protease. Nature 1989, 339:191-195.
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FOTHERGILL MD, FERSHT AR: Correlations Between Kinetic and X-Ray Analyses of Engineered Enzymes: Crystal Structures of Mutants Cys --+ Gly-35 and Tyr ~ Phe-34 of Tyrosyl-tRNA Synthetase. Biochemistry 1991, 30:5157-5164.
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CHOTHIA C, LESK AM, DODSON GG, HODGKIN DC: Transmission of Conformational Change in Insulin. Nature 1983, 302:500-505.
6. MATII-mWSBW: Crystallography in the Life Sciences. Cry~ ,° tallogr Rev 1990, 72:133>162. A review of recent advances in protein crystallography that is of p~articular interest to non-specialists. The work is well illustrated and gives a particularly good brief account of the very important work of the au thor. MATrHEWS BW: Mutational Analysis of Protein Stability. Curt Opin Struct Biol 1991, 1:17-21. REIDHARR-OiSONJV, SAUERRT: Combinatorial Cassette Mutagenesis as a Probe of t h e Informational C o n t e n t of Protein Sequences. Science 1988, 241:53-57. ElM WA~ SAUERRT: Alternative Packing Arrangements in t h e Hydrophobic Core of ~, Repressor. Nature 1989, 339:3106. 10. ,•
LIM WA, SAUERRT: The Role of Internal Packing Interactions in Determining the Structure and Stability of a Protein. J Mol Biol 1991, 219:359-376. Five amino acids in the hydrophobic core of the amino-terminal domain of ~L-repressor were randomly mutated to residues from the set of large hydrophobics, valine, leucine, isoleucine, methionine and phenylalanine, in an effort to explore systematically the requirements for folding and function. About 70% of the 78 molecules isolated were functional to some extent.
11.
BOWIEJU, REIDHARR-OLSONJF, LIM WA, SALTERRT: Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions. Science 1990, 247:1306-1310. Families of sequences compatible with a particular protein fold are examined to show what features must be retained, and what degree of variability is tolerable. .
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REIDHAAR-OLSONJF, SAUER RT: Functionally Acceptable Substitutions in Two s-Helical Regions of ~LRepressor. Proteins 1990, 7:306-310.
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PERUTZMF (ED): M e c h a n i s m s of Cooperativity and Allosteric Regulation in Proteins [book]. Cambridge University Press, 1990. A detailed, thorough and profound review of structural changes in proteins.
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LESKAM, CHOTHIA C: HOW Different Amino Acid Sequences Determine Similar Protein Structures: t h e Structure and Evolutionary Dynamics of t h e Globins. J Mol Biol 1980, 136:225-270.
PASTORE A~ LESK AM: Comparison of t h e Structures of Globins and Phycocyanins: Evidence for Evolutionary Relationship. Proteins 1990, 8:133>155. This paper addresses the question of h o w to distinguish homology from convergent evolution in proteins between which no relationship can be detected in the sequences. It is shown that phycocyanin and the globins share many structural details that would not be expected to exist if they arose independently.
34. •
BOWIEJU, CLARKE ND, PABO CO, SAUER RT: Identification of Protein Folds: Matching Hydrophobicity Patterns of Seq u e n c e Sets with Solvent Accessibility Patterns of K n o w n Structures. Proteins 1990, 7:257-264. Given a set of related protein sequences, the pattem of hydrophobicity is correlated with positions of residues on the protein surface, and can characterize a folding pattern well enough to associate a sequence with the correct tertiary fold from a data bank of known structures. 35.
OVERINGTON J, JOHNSON MS, SALI A, BLUNDELL TL: Tertiary Structural Constraints o n Protein Evolutionary Diversity: Templates, Key Residues and Structural Prediction. Proc Roy Soc Lond B 1990, 241:132-145.
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A Pastore, EMBL, Meyerhofstrasse 1, 6900 Heidelberg, Germany. AM Lesk, Department of Haematology, University of Cambridge Clinical School, MRC Centre, Hills Road, Cambridge CB2 2QH, UK.
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are accidents of history. This situation has implications for biotechnology. In order to decide which structural features might be reasonable design targets for synthetic proteins, it is important to try to understand the constraints imposed by natural evolution and to recognize historical accidents (to avoid being "condemned to repeat them"). Conversely, the synthesis and assessment of the structural and functional properties of non-natural proteins can help to distinguish physical law from historical accident.
Natural molecular evolution Studies of natural protein evolution have shown that, as amino acid sequences diverge by mutations, insertions and deletions, the protein conformation changes [1]. The larger the divergence of the sequences, the larger the distortion of the conformations. Several observations have been made about the divergence of homologous proteins during evolution [2,3]. First, a core of the secondary structure, including the active site, retains its folding pattern. Other regions of the molecule may refold entirely. Second, although individual helices or sheets within the core tend to retain standard conformations, they shift and rotate with, respect to one another. This happens because stability of the protein requires formation of well packed interfaces between elements of secondary structure. When mutations alter residues at interfaces, the packing must adjust. Typically, there is room to accommodate the extra odd methyl group without significantly changing the main-chain conformation. But (for example) a substantial increase in side-chain volume at an interface will tend to push the helices or sheets apart. A third observation is that the pattern of residue-residue contacts at interfaces tends to be preserved. This is consistent with evolutionary pathways in which substitution of one residue at a time allows the retention of a well packed interface by adjustment of the relative geometry of the interacting helices or sheets. The pattern of
(~ Current Biology Ltd ISSN 0958-1669
Brave new proteins: what evolution reveals about protein structure Pastore and Lesk S93 residue-residue interactions at an interface influences the relative geometry of the elements of seconda~ structure. For packed helices, for example, the structural pattern at the interface is correlated with a range of interaxial angles. This suggests that the conservation of the pattern of residue interactions is not merely evolutionary stodginess but may be essential either for the stability of the native state or possibly for the folding pathway. Fig. 1 summarizes schematically the progressive divergence of sequences within a family of proteins. Amino acid sequence similarity, measured by the number of identical residues in the correct sequence alignment of a pair of proteins, decreases from 100% on the left to below the noise level on the right. Down to about 40-50% sequence identity the structures are very similar. From 20-40% sequence identity, the basic fold of the core of a protein family has a conserved topology but the structures are distorted. Below 20%, one cannot be sure from sequences alone that two proteins are homologous: the fold may well be conserved although the homology cannot be recognized in the sequences. That is, only proteins with above 20% sequence identity can be identified as homologous, but proteins with below 20% sequence identity cannot be assumed not to be homologous.
Proteins definitely homologo Structures very similar _< 1.0 ~ root mean square main-chain deviation
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0 % Identical amino acids
Fig. 1. The relationship between divergence of amino acid sequence and divergence of structure in proteins. For simplicity, we consider three classes of closeness of relationship (the boundaries are only approximate): from 100% residue identity down to 50%, protein structures are very similar; from 50% to 25% residue identity, the proteins are definitely homologous but their structures show substantial differences; below 25%, proteins may be homologous and similar in structure or may be unrelated.
Very distantly related proteins often arise through evolution with change of function. During evolutionary events that procede with retention of function, global constraints on the structure of a protein link the geometric changes at interfaces between elements of secondary structure [ 1]. In evolution with change in function, these constraints are released, or, rather, replaced by other constraints. Two sequences can diverge to the point at which the relationship between them cannot be recognized, and the structural changes are much larger than in closely related proteins. The crystallins provide a good example of this process at work. A variety of enzymes have been recruited for structural roles in lenses of ver-
tebrates; some but not all of them retain their enzymatic av ....
u~-,.
[qj.
Synthetic proteins Synthetic single-site mutations can be carried out to explore novel sequences in a way comparable to evolution. (For reviews see [5,6", 7]). In contrast, multiple simultaneous amino acid substitutions allow the exploration of sequences inaccessible to natural evolution. There have been a number of studies of multiple synthetic mutants, notably by Sauer and coworkers [8,9,10",11"-13"]. On the computational side, Lee and Levitt [14,] have been able to predict the relative stabilities of some of these mutants. Ponder and Richards [15] have written programs to search for alternative packings of a hydrophobic core, and applications of this program to synthetic work have begun.
Structural studies of synthetically mutated proteins X-ray crystallography makes it possible to describe in detail the structural consequences of mutations and to correlate them with changes in stability and function. The combination of mutagenesis and structure determination makes it possible to probe selected structural features systematically. Areas of recent progress include (but are not limited to): the quality of hydrophobic packing in protein interiors and its effect on stability; addition or deletion of disulfide bridges; and changes in function associated with mutations at active sites. Matthews and coworkers [16-20] have made mutants of T4 lysozyme and solved many of them to high resolution. To explore the effect of quality of packing in the interior, they increased the size of a side chain adjacent to either of the two largest cavities within the structure. The two mutants, lmu133Phe and Ala139Val, had normal activity but slightly reduced stability (melting temperature lowered by 1-3°C). The structural perturbations were small (largest shift about 0.7g,) and localized near the site of mutation; in the Ala139Val mutant a portion of an a-helix shifts concertedly by 0.15g~ In each case, the mutant side chain is somewhat strained, accepting non-ideal sidechain torsional angles and mildly uncomfortable steric interactions, thus offsetting the gain in stability arising from burying additional non-polar atoms. It is interesting that the structure is only partially able to 'relax' around the mutations. In related work, the structures of two T4 lysozyme mutants Ile3Val and Ile3Tyr were examined [ 17]. In the wildtype, Ile3 sits in a hydrophobic pocket; only its C2-2 atom is solvent-accessible. Reducing the side chain volume in Ile3Val produced a structure 'essentially identical to the wild-type'. In contrast, the tyrosine side chain at this site
594
Proteinengineering does not fit into the pocket, and turns out into the solvent, a water molecule entering the cavity left behind. Conformational changes include shifts in side chains adjacent to the site of mutation and concerted shifts in two helices, by about 0.6-1.0A. Structural studies of the consequences of additions and deletions of disulfide bridges have been carried out in T4 lysozyme [18-20], bovine pancreatic trypsin inhibitor [21] and subtilisin BPN' [22]. Disulfide bridges inserted into T4 lysozyme at residue pairs 9--164, 21-142 and 3-97 increase stability, additively; the triple disulfide mutant has a melting temperature 23.4°C higher than the wildtype. Removal of disulfide bridge 30-51 in bovine pancreatic trypsin inhibitor by changing both cysteines to alanines showed localized shifts in two segments of chain (about five residues surrounding each site of mutation) to try to fill the gap [21]. The structural analysis of functional changes arising from mutations of active-site residues is seen in mutants of a-lytic protease [23] and tyrosyl-tRNA synthetase [24]. Alpha-lytic protease is a serine protease with a specificity for alanine in the P1 site, which is produced by occupancy of the specificity pocket by the side chain of Met192. The mutants Metl9ZAla and Met213Ala (Met213 is adjacent to the specificity pocket) showed broadened specificity, arising from removal of the large side chain and small changes in and around the binding pocket. Confonnational changes were observed, both with and without ligand, an-d it was pointed out that the ability of the structure to deform to accommodate ligands enhanced the range of specificity. What can we learn from these structures? We know that proteins are somewhat plastic, from comparisons of structures of proteins in different crystal forms, which show the deformations induced by different crystal packing forces [2,3]. Indeed, Eigenbrot, Randall and Kossiakoff [21] point out that the structural changes observed in mutant proteins "are of the same order of magnitude or smaller than those associated with the lattice forces alone". In general, in response to a mutation, the structure tries to deform to compensate. Of course, this cannot always be carried out successfully. The repertoire of available responses includes local deformation and binding of solvent in cavities created. Several authors [18-20,22] have noted concerted shifts in a-helices, which have also been observed in response to crystal packing forces and in conformational changes arising from changes in ligation states [25-27]. Thus, the capacity of proteins for structural change is important not only for permitting evolution to explore sequence changes, but also for functional applications, for example in enzymes that show 'induced fit' or allosteric changes (recently reviewed by Perutz [28-,]).
Multiple mutagenesis as a probe of sequences compatible with a fold Lim and Sauer [9,10"] have used cassette mutagenesis to randomize selected sets of seven residues in the hydrophobic core of the amino-terminal domain of phage £-repressor limiting their substitutions to the large hydrophobic residues valine, leucine, isoleucine, methionine and phenylalanine. Selecting for functional molecules followed by sequencing produced a catalogue of sequences that are compatible with the native fold and showed that most form reasonably stable structures although, in many cases, with reduced activity. Several properties of the core residues essential for folding were noted, including retention of hydrophobicity. A restraint in total volume changes was also observed: the total volume of the core residues was found to lie within the equivalent of two extra or three fewer methylene groups of the wild-type. Finally, a 'steric constraint' was found to exist, in that different sequences with similar core hydrophobicity and total side-chain volume were not always functionally equivalent. That is, the requirements for function are more stringent than the requirements for the general overall folding pattern. For example, three residues in the core contain, in the original protein, Va136, Met40 and Val47. The single mutant Met40Leu is functional but the double mutant of the same amino acid composition Va136Leu/Met40Val is only partially functional. The authors interpret these differences in terms of the steric quality of the packing of these residues. Clearly the structural shifts produced by the different packings are affecting the relative spatial disposition of groups in these proteins important for function. Other studies by this group based on the same technique include the exploration of acceptable substitutions in a helix interface in the ~v-repressor [12.], and in a molecule containing a leucine zipper, constructed by fusing the GCN4 leucine zipper domain with the £-repressor. The role of the repressor in these experiments is primarily to assay for dimerization of the leucine zipper domain [13"].
Structural implications of packing constraints Lacking the structures of the synthetic multiple mutants of the ~,-repressor, we illustrate steric constraints on packing in natural proteins. Chothia (see [29]) showed that interfaces between packed helices in protein interiors are not merely 'hydrophobic glue' but have specific structures. The side chains on the surfaces of the helices form well defined ridges and grooves, and interfaces form by packing ridges on the surface of one helix into grooves of the surface of the other. There are three main ways of forming ridges on a helix surface, and their interactions give rise to distinct classes of interface patterns, with typical interaxial angles.
Brave new proteins: what evolution reveals about protein structure Pastore and Lesk 595
(b)
.
,
Fig. 2. Van der Waals slices showing (a) the packing at the interface between B and G helices in sperm whale myoglobin, showing the most common type of 'ridges-into-grooves' packing and (b) the packing at the interface between B and E helices in sperm whale myoglobin, showing the unusual 'crossedridge' structure of the interface, in which ridges from the two helices cross over a 'notch'. Residues from the B helix are shown as solid lines; those from the G helix (a) and E helix (b) are shown as broken lines. Thick lines indicate ridges. Two examples illustrate the structural specificity of the packing [30]. Fig. 2(a) shows the interface between the B and G helices of sperm whale myoglobin, illustrating the most common type of 'ridges-into-grooves' packing. In contrast, Fig. 2(b) shows the interface between the B and E helices of myoglobin, which has an unusual 'crossed-ridge' structure, a packing pattern in which ridges from the two helices cross over a 'notch'. The ridges are formed by the residues Va121, Gly25, Leu29 and Phe33 (from the B helix) and Leu61, Gly65 and Leu69 (from the E helix). Each glycine residue forms a notcl'f in its ridge. The two glycine residues sit over each other, allowing the ridges to cross without loss of good packing. This structure imposes a constraint on the amino acid sequences, in terms of the relative volumes of adjacent residues on the helix surface. Studies of homologous proteins show that as protein families diverge, the class of the packing is retained. This is shown in Fig. 3, for the helix contacts in the globins. This conservation also extends to phycocyanin [31o.].
Role of packing in determining structure Behe et al. [32] have argued recently that packing does not determine the native fold. Although there is a consensus that stabilization of the native state requires efficient packing of hydrophobic residues in the protein interior, the authors suggest that these interactions have little specificity, and that many combinations of hydrophobic residues could form well packed interiors of comparable stability. Given the extreme form of this premise that quality of packing is sequence independent, then provided buried residues remain hydrophobic, it would follow that the three-dimensional structure cannot be determined by the identities of the residues at the buried sites;
Behe et al. [32] have presented statistical data on the interactions of residue pairs in natural proteins of known structure. They were looking for two types of relationships, the first being preferred pairwise complementarity of side chains, that is, residues that, because of mutual conformability, are preferentially in contact within protein interiors (e.g."... leucine residues might nestle together like spoons ..." [32]), and the second being preferred pairs of residues that pack unusually efficiently with their surroundings. No such preferences, in either case, were found. In the context of this review, it should be noted that Behe et al. are in fact discussing native proteins, and not the potentially more general sets of sequences that might produce the same fold but are inaccessible to natural evolution. Their statistical analysis of residue interactions is based on crystal structures of natural protein structures. Their suggestion that it is patterns of hydrophobicity that define conformation is based on the feasibility of classifying natural sequences on the basis of hydrophobicity pattems [33,34"]. Behe et al. conclude that specificity in residue packing cannot determine protein conformation. In other words, packing interactions do not constrain the sequences that will produce a given folding pattern. In natural proteins, however, there are structural constraints on protein interiors (Fig. 2b), in which the relative volumes of residues create the notch that permits the conserved crossed-ridge structure. Comparisons of related structures show that the conservation patterns are considerably more subtle than pairwise correlations of residues (except for disulfide bridges) [35]. Analyses of aligned sequences for patterns of hydrophobicity that define folding pattems do, in fact, include restraints or even constraints on residue volumes at certain positions [33,34"]. For example, Bowie et al. [34"] show that patterns derived from aligned sequences form identifying 'signatures' of folding pattems for globins, cy-
596
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f~ Oo
12
10 ~J
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I
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tochromes c, CheY and calcium-binding proteins. Their patterns represent residues by a three-letter 'alphabet' corresponding to classes of hydrophobicity according to the scale of Fauchere and Pliska [36]. The three classes of residues are WIPI_MVC, YPATHGS and QNEDKR (residues identified by one-letter codes). In fact, these classes do segregate residues by volume as well as by charge or polarity. The structural constraints observed in natural molecules do not preclude the possibility of other modes of packing consistent with a given fold, which might accommodate a wider range of sequences. These might arise in ge netic engineering experiments such as those of Sauer and coworkers [8,9,10oo,11.-13°], or be suggested by calculations of the type of Ponder and Richards [15]. The specificity in packing interactions that are observed in known structures may be partly historical accidents, and other structures, showing less specificity, may well be stable. An example is discussed in the next section. Until such possibilities are more fully explored, however, it would seem premature to exclude packing constraints from any possible role in determining folding patterns.
Non-natural globins: a challenge It is possible to detect, in Fig. 3, an opportunity to create a structure that might be functional, but not accessible to evolution from known natural globins. Although
Fig. 3. The distribution of interaxial distances and angles in the major helix contacts in the globins. Homologous pairs of helices retain the structure of the interface, in terms of the pattern of residue packing. Letters signify pairs of packed helices - - for example, B-G indicates the interaction between the B and G helices. Symbols at large dots indicate the species of origin of the globin. Ha, human haemoglobin a-chain; H~, human haemoglobin 1[3chain; Ea, horse haemoglobin a-chain; E~, horse haemoglobin [[]-chain; W, sperm whale myoglobin; L, sea lamprey globin; G, Glycera globin; C, Chironomus erythrocruorin; Lg, lupin leghaemoglobin [15].
homologous helix interfaces cluster in interaxial distance and angle, the B-E and B-G classes, which have different modes of residue packing, approach each other. For example, the B-E interface of Glycera globin has an interaxial distance and angle very similar to that of the B-G contact in Cbironomus erythrocruorin, although the pattern of residue interactions is different. This suggests that it would be interesting to try to transfer the interface; that is, to create a non-natural globin by modifying (for example) Glycera globin with multiple simultaneous mutations so that the B-E interface contains the residues and the packing of the B--G interface of Chironomus erythrocruorin, or perhaps some other such pair that appears to be the most compatible structurally. (This is an example of the kind of alternative packings that the methods of Ponder and Richards [15] are designed to detect.) If this experiment were successful it would produce a globin that, as far as we know, is inaccessible to natural evolution.
Conclusion Natural protein evolution imposes constraints on protein structures that do not apply to molecules created by genetic engineering. An understanding of such constraints can suggest what kinds of structures might be achievable synthetically. The creation of non-natural structures is necessary to illuminate the principles that underly the features of natural ones.
Brave new proteins: what evolution reveals about protein structure Pastore and kesk
Acknowledgements We both thank C Chothia and AG Murzin for helpful comments. AM Lesk also thanks the Kay Kendall Foundation for generous support.
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By random mutagenesis the sequence requirments of 25 positions in the amino-terminal domain of ~.-repressor are investigated. A variety of degrees of tolerance was observed. O n the suface, s o m e positions are very robust and will accept essentially all types of residues. Others accept only hydrophilic residues and one requires proline. Buried residues are constrained relatively stringently. 13. •
Hu JC, O'SHEA EK, KIM PS, SAUERRT: Sequence Requirements for Coiled-Coils: Analysis w i t h ~ Repressor--GCN4 Leucine Zipper Fusions. Science 1990, 250:1400-1403. Eight positions in the hydrophobic interface of the leucine zipper dimer from GCN4 were subjected to mutation, in order to determine the acceptable range of variability at these sites. Most single mutants that retained hydrophic character, but not all multiple mutants were functional. Leucine was not essential at the special positions but was favoured. LEE C, LEVITt M: Accurate Predication of t h e Stability and Activity Effects of Site-Directed Mutagenesis o n a Protein Core. Nature 1991, 352:448-450. Stabilization energies of 78 triple mutants of X-repressor have been calculated and are well correlated with the thermostabilities of the mutants. Active and inactive mutants are discriminated with 92% accuracy. 14. .
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6. MATII-mWSBW: Crystallography in the Life Sciences. Cry~ ,° tallogr Rev 1990, 72:133>162. A review of recent advances in protein crystallography that is of p~articular interest to non-specialists. The work is well illustrated and gives a particularly good brief account of the very important work of the au thor. MATrHEWS BW: Mutational Analysis of Protein Stability. Curt Opin Struct Biol 1991, 1:17-21. REIDHARR-OiSONJV, SAUERRT: Combinatorial Cassette Mutagenesis as a Probe of t h e Informational C o n t e n t of Protein Sequences. Science 1988, 241:53-57. ElM WA~ SAUERRT: Alternative Packing Arrangements in t h e Hydrophobic Core of ~, Repressor. Nature 1989, 339:3106. 10. ,•
LIM WA, SAUERRT: The Role of Internal Packing Interactions in Determining the Structure and Stability of a Protein. J Mol Biol 1991, 219:359-376. Five amino acids in the hydrophobic core of the amino-terminal domain of ~L-repressor were randomly mutated to residues from the set of large hydrophobics, valine, leucine, isoleucine, methionine and phenylalanine, in an effort to explore systematically the requirements for folding and function. About 70% of the 78 molecules isolated were functional to some extent.
11.
BOWIEJU, REIDHARR-OLSONJF, LIM WA, SALTERRT: Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions. Science 1990, 247:1306-1310. Families of sequences compatible with a particular protein fold are examined to show what features must be retained, and what degree of variability is tolerable. .
12. .
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34. •
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A Pastore, EMBL, Meyerhofstrasse 1, 6900 Heidelberg, Germany. AM Lesk, Department of Haematology, University of Cambridge Clinical School, MRC Centre, Hills Road, Cambridge CB2 2QH, UK.
