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Mutational Effects on Stability Are Largely Conserved During Protein Evolution

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Mutational Effects on Stability Are Largely Conserved During Protein Evolution Mutational effects on stability are largely conserved during protein evolution Orr Ashenberg1, L. Ian Gong1, and Jesse D. Bloom2 Division of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109 Edited by Andrew J. Roger, Centre for Comparative Genomics and Evolutionary Bioinformatics, Dalhousie University, Halifax, Canada, and accepted bythe Editorial Board November 15, 2013 (received for review August 5, 2013) Protein stability and folding are the result of cooperative inter- approaches used to model protein evolution? The standard way actions among many residues, yet phylogenetic approaches to address this question has been to simulate or analyze protein assume that sites are independent. This discrepancy has engen- sequences, using some computational force field that predicts the dered concerns about large evolutionary shifts in mutational stability of different variants (2–5). Unfortunately, there is no effects that might confound phylogenetic approaches. Here we good a priori reason to believe that the force fields themselves experimentally investigate this issue by introducing the same authentically represent interactions among sites. Even state-of- fl mutations into a set of diverged homologs of the in uenza the-art force fields are at best modestly accurate at predicting fi nucleoprotein and measuring the effects on stability. We nd that mutational effects (6, 7). Furthermore, all tractable force fields mutational effects on stability are largely conserved across the approximate the true multibody quantum–mechanical interactions homologs. We reach qualitatively similar conclusions when we – simulate protein evolution with molecular-mechanics force fields. among protein sites in terms of pairwise interactions (8 10). Our results do not mean that proteins evolve without epistasis, Here we experimentally assess the extent to which mutational which can still arise even when mutational stability effects are effects on stability change during protein evolution. We in- conserved. However, our findings indicate that large evolutionary troduce the same mutations into a series of homologs and shifts in mutational effects on stability are rare, at least among measure the effects on stability. We find that the effects of in- homologs with similar structures and functions. We suggest that dividual mutations on stability are largely conserved even when properly describing the clearly observable and highly conserved homologs differ at as many as 28% of sites. Our results do not amino acid preferences at individual sites is likely to be far more imply an absence of all forms of epistasis (interactions among important for phylogenetic analyses than accounting for rare sites) during protein evolution. Epistasis at the level of function shifts in amino acid propensities due to site covariation. and fitness can arise even if stability is fully site independent due to counterbalancing stabilizing and destabilizing mutations (11, heterotachy | consensus design | substitution models 12), and stability is only one mechanism of epistasis (13, 14). However, for reasons that we discuss below, these other forms of ost biological proteins stably fold to a well-defined native epistasis seem less likely to cause pervasive shifts in substitution Mstructure (1). Stable folding to the native structure is typ- patterns that would seriously confound phylogenetic approaches. ically essential for a protein to perform its evolutionarily selected However, our experiments do highlight the existence of strong function. Stability and folding are the result of highly cooperative and largely conserved preferences for certain amino acids at ’ interactions among a protein s constituent amino acids (1). specific sites. We therefore suggest that the use and further de- Therefore, evolutionary selection at a site in a protein acts on velopment of phylogenetic approaches (15–24) that account for properties that in principle are determined by interactions of that these clearly observable site-specific but largely site-independent site with every other position in the sequence. However, phylogenetic approaches assume that protein sites Significance evolve independently. This assumption has historically been justified primarily by pragmatic considerations. If substitution models are site independent, then the evolution of each site is The properties of proteins are in principle determined by mathematically described by a 20 × 20 matrix, with entries cor- interactions among all of their residues, but for practical rea- responding to all amino acid interchanges at that site. On the sons, phylogenetic analyses assume that residues change in- other hand, if each site depends upon all other sites, then the dependently. We experimentally investigate the extent to evolution of a protein of length L is mathematically described which the effects of individual mutations on protein stability L L change as other residues diverge. We find that mutational by a 20 × 20 matrix, with entries corresponding to all inter- L effects on stability are highly conserved, with relatively little changes among the 20 sequences. Even for a small protein of interdependence. Of course, mutations can still interact in length L = 100, the number of entries in such a matrix vastly other ways to affect protein function in a nonindependent exceeds the number of atoms in the universe, which poses severe manner. Nonetheless, our results suggest that the preference computational challenges. of a position for specific amino acids tends to be well con- The fact that sites are not actually independent at the physi- served during the evolution of protein homologs with similar cochemical level has provoked concern about the use of site- structures and functions. independent substitution models (2–5). Pollock et al. (2) have used computer simulations to suggest that there are widespread Author contributions: O.A. and J.D.B. designed research; O.A. and L.I.G. performed re- evolutionary shifts in mutational effects, where for instance search; O.A., L.I.G., and J.D.B. analyzed data; and O.A., L.I.G., and J.D.B. wrote the paper. a mutation is destabilizing in one homolog but stabilizing in The authors declare no conflict of interest. another homolog due to interactions with other covarying sites. This article is a PNAS Direct Submission. A.J.R. is a guest editor invited by the Editorial They have argued that such evolutionary shifts in mutational Board. effects on stability have profound implications for phylogenetic 1O.A. and L.I.G. contributed equally to this work. modeling (2). 2To whom correspondence should be addressed. E-mail: [email protected]. fi EVOLUTION However, are such evolutionary shifts really of suf cient This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. prevalence and magnitude to require a radical rethinking of 1073/pnas.1314781111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1314781111 PNAS | December 24, 2013 | vol. 110 | no. 52 | 21071–21076 Downloaded by guest on September 26, 2021 preferences is of far greater practical importance than account- our previous work (11), we expected that in Brisbane/2007, the ing for rare evolutionary shifts in site preferences. first two mutations would be stabilizing, the next two would have little effect, and the last two would be destabilizing. Fig. 1B Results shows the mutations in the NP structure, as well as the wild-type Analysis of Mutational Effects in a Set of Homologs. We introduced identities in each homolog. Fig. 1B also shows all other sites that the same mutations into a set of homologs of the nucleoprotein differ between Brisbane/2007 and the other homologs. The (NP) of influenza A virus. NP is a 498-residue protein that serves protein-sequence identities of the homologs with Brisbane/2007 as a scaffold for viral RNA during replication, transcription, and range from 94% to 72%. The number of substitutions that oc- genome packaging (25). Crystal structures of several NPs have curred during divergence of the homologs is not an observable been solved (26–28), and all fold to similar mostly alpha-helical quantity. However, inferences made using PAML (31) with two conformations. NP multimerizes, but we have developed a pro- common substitution models (32, 33) suggest that the most distant tocol for purifying monomeric NP and measuring its stability (11). pair diverged via 200- to 300-amino-acid substitutions (Table S1). As the reference protein for our study, we chose the NP of a recent human H3N2 strain, A/Brisbane/10/2007. We then se- Experimentally Measured Mutational Effects on Stability Are Largely lected NPs from three increasingly diverged strains: the human Conserved. We cloned and purified histidine-tagged variants of H3N2 strain A/Aichi/2/1968, the human 2009 H1N1 pandemic each homolog. Fig. S1 shows that all variants exhibited similar strain A/California/4/2009, and the bat strain A/little yellow- circular dichroism (CD) spectra with the classical alpha-helical shouldered bat/Guatemala/164/2009. Fig. 1A shows the relation- minima near 208 and 222 nm (34), suggesting that all variants ship among these NPs. Brisbane/2007, Aichi/1968, and California/ retained the alpha-helical NP structure (26–28). The only variant 2009 NPs are descended from a close predecessor of the 1918 that we could not purify was bat/2009 with L259S; we suspect pandemic virus. The bat/2009 NP is highly diverged from all other that this variant is too destabilized to yield folded protein (Fig.
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