Systematic Variation of Amino Acid Substitutions for Stringent Assessment of Pairwise Covariation

Govindarajan, Sridhar; Ness, Jon E.; Kim, Seran; Mundorff, Emily C.; Minshull, Jeremy; Gustafsson, Claes

doi:10.1016/s0022-2836(03)00357-7

Cited by 41 publications

(24 citation statements)

References 32 publications

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“…Improving these approaches will require the ability to successfully capture the context-dependent effects we observed, and our study therefore provides a valuable benchmark for testing the accuracy and sensitivity of protein modeling and design methods. Context-dependent effects of mutations (epistasis) have been characterized both within single proteins (20)(21)(22) and in proteinprotein interfaces (23,24). In general, epistatic effects within proteins, although likely important on evolutionary timescales, have been suggested to be rare individually as the majority of pairwise mutations appear compatible (20,21).…”

Section: Discussionmentioning

confidence: 99%

“…Context-dependent effects of mutations (epistasis) have been characterized both within single proteins (20)(21)(22) and in proteinprotein interfaces (23,24). In general, epistatic effects within proteins, although likely important on evolutionary timescales, have been suggested to be rare individually as the majority of pairwise mutations appear compatible (20,21). Similarly, in protein-protein interfaces, pairs of mutations have been found to have largely additive energetic effects unless the mutated residues are in direct contact (23) or part of the same tightly interacting cluster (25).…”

Section: Discussionmentioning

confidence: 99%

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Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition

Melero

Ollikainen

Harwood

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Reengineering protein-protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of "second-site suppressors," where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein-protein interfaces. To extend this approach, it would be advantageous to be able to "transplant" existing engineered and experimentally validated specificity changes to other homologous protein-protein complexes. Here, we test this strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain-peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein-protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. Although the context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein-protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context. computational design | recognition specificity | promiscuity | protein interaction domains | interface evolution M any protein-protein interactions are mediated by small modular protein recognition domains (1). These interaction modules, such as PDZ, SH3, and WW domains, generally recognize their protein partners using a structurally conserved binding site, and there are often tens or even hundreds of proteins containing a given type of recognition domain expressed simultaneously in a cell or organism (2). This repeated use of recognition modules with conserved structures poses the following question: how do cells maintain the specificity of binding interactions when so many members of the same domain family are present? Moreover, to what extent do different domain family members in fact have distinct or overlapping preferences for binding their partners? Addressing these questions is of considerable importance, because a significant fraction of protein interactions in a cell is mediated a limited number of protein interaction domain families (1). Despite a large amount of information on the biochemical recognition preferences of many domain members in vitro (3-5), much less is known about the actual extent of specificity an...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition

Melero

Ollikainen

Harwood

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…nature of interacting amino acid residues. Such interdependence of physical interactions seems bound to lead to interdependence, or coevolution, in the evolutionary process, and coevolution has indeed been detected on numerous occasions Chelvanayagam et al, 1997;Fukami-Kobayashi et al, 2002;Gobel et al, 1994;Govindarajan et al, 2003;Korber et al, 1993;Lapedes et al, 1997;Neher 1994;Pazos et al, 1997;Pollock and Taylor, 1997;Pollock et al, 1999;Pritchard et al, 2001;Shindyalov et al, 1994;Taylor and Hatrick, 1994;Tuff and Darlu, 2000;Valencia and Pazos, 2002;Wollenberg and Atchley, 2000). Interdependence should also lead to changes in rates at individual sites during the normal course of evolution, and such rate changes have been found to occur regularly in the absence of functional change Lopez et al, 2002;Philippe et al, 2003), sending a loud warning to those who would define functional divergence as synonymous with rate change.…”

Section: Introductionmentioning

confidence: 99%

Context Dependence and Coevolution Among Amino Acid Residues in Proteins

Wang¹,

Pollock²

2005

Methods in Enzymology

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As complete genomes accumulate and the generation of genomic biodiversity proceeds at an accelerating pace, the need to understand the interaction between sequence evolution and protein structure and function rises in prominence. The pattern and pace of substitutions in proteins can provide important clues to functional importance, functional divergence, and adaptive response. Coevolution between amino acid residues and the context dependence of the evolutionary process are often ignored, however, because of their complexity, but they are critical for the accurate interpretation of reconstructed evolutionary events. Because residues interact with one another, and because the effect of substitutions can depend on the structural and physiological environment in which they occur, an accurate science of evolutionary functional genomics and a complete understanding of selection in proteins require a better understanding of how context dependence affects protein evolution. Here, we present new evidence from vertebrate cytochrome oxidase sequences that pairwise coevolutionary interactions between protein residues are highly dependent on tertiary and secondary structure. We also discuss theoretical predictions that impinge on our expectations of how protein residues may interact over long distances because of their shared need to maintain protein stability.

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“…These correlation signals are stronger when obtaining measurements using ancestral sequences inferred from phylogenetic data (27,28). In a similar effort, Govindarajan et al (29) showed that for many pairs of positions in protein families certain residue combinations are highly preferred. It is reasonable to expect that the same correlation pattern may extend to the properties of specific residue pairs, e.g., size, hydrophobicity, and charge (30).…”

mentioning

confidence: 99%

FamClash: A method for ranking the activity of engineered enzymes

Saraf

Horswill

Benkovic

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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This article introduces the computational procedure FamClash for analyzing incompatibilities in engineered protein hybrids by using protein family sequence data. All pairs of residue positions in the sequence alignment that conserve the property triplet of charge, volume, and hydrophobicity are first identified, and significant deviations are denoted as residue-residue clashes. This approach moves beyond earlier efforts aimed at solely classifying hybrids as functional or nonfunctional by correlating the rank ordering of these hybrids based on their activity levels. Experimental testing of this approach was performed in parallel to assess the predictive ability of FamClash. As a model system, single-crossover ITCHY (incremental truncation for the creation of hybrid enzymes) libraries were prepared from the Escherichia coli and Bacillus subtilis dihydrofolate reductases, and the activities of functional hybrids were determined. Comparisons of the predicted clash map as a function of crossover position revealed good agreement with activity data, reproducing the observed V shape and matching the location of a local peak in activity.protein engineering ͉ dihydrofolate reductase ͉ residue-residue clash ͉ computational hybrid prescreening ͉ incremental truncation R ecent advances in protein engineering (1-5) have allowed researchers to go beyond the limitations of homologydependent directed evolution methods. The ability to freely explore protein sequence space has revealed a number of troublesome trends. First, the lower the sequence identity of the recombined parental sequences, the smaller the percentage of the combinatorial protein library that remains functional (2, 4). This has been reported in several studies (6-8) using differing protocols, thus implicating the global nature of this effect. More troublesome is the finding that the remaining functional hybrids tend to have only residual activities. Therefore, it appears that exploring protein sequence space freely comes at the expense of severely degrading the average stability and functionality of the combinatorial library. This has motivated the development of computational methods to prescreen hybrids for their potential of being stably folded (9) and functional. These analyses then serve to direct the sampling of protein sequences by the combinatorial library toward desirable regions in sequence space. Specifically, favorable positions for junctions between fragments from different parental sequences can be identified, and restrictions can be imposed on sets of parental sequences that contribute fragments to a particular junction.Therefore, further improvements in the stability and functionality of hybrid proteins may be attained by developing quantitative methods that identify deleterious interactions arising from residue pairs within the gene fragment combinations. To this end, Monte Carlo simulations by Bogarad and Deem (10) suggested that swapping of low-energy structures is least disruptive to protein structure. The SCHEMA algorithm (11) postulates t...

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Systematic Variation of Amino Acid Substitutions for Stringent Assessment of Pairwise Covariation

Cited by 41 publications

References 32 publications

Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition

Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition

Context Dependence and Coevolution Among Amino Acid Residues in Proteins

FamClash: A method for ranking the activity of engineered enzymes

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