Amino acid positions subject to multiple co-evolutionary constraints can be robustly identified by their eigenvector network centrality scores

As proteins evolve, amino acid positions key to protein structure or function are subject to mutational constraints. These positions can be detected by analyzing sequence families for amino acid conservation or for co-evolution between pairs of positions. Co-evolutionary scores are usually rank-ordered and thresholded to reveal the top pairwise scores, but they also can be treated as weighted networks. Here, we used network analyses to bypass a major complication of co-evolution studies: For a given sequence alignment, alternative algorithms usually identify different, top pairwise scores. We reconciled results from five commonly-used, mathematically divergent algorithms (ELSC, McBASC, OMES, SCA, and ZNMI), using the LacI/GalR and 1,6-bisphosphate aldolase protein families as models. Calculations used unthresholded co-evolution scores from which column-specific properties such as sequence entropy and random noise were subtracted; “central” positions were identified by calculating various network centrality scores. When compared among algorithms, network centrality methods, particularly eigenvector centrality, showed markedly better agreement than comparisons of the top pairwise scores. Positions with large centrality scores occurred at key structural locations and/or were functionally sensitive to mutations. Further, the top central positions often differed from those with top pairwise co-evolution scores: Instead of a few strong scores, central positions often had multiple, moderate scores. We conclude that eigenvector centrality calculations reveal a robust evolutionary pattern of constraints – detectable by divergent algorithms – that occur at key protein locations. Finally, we discuss the fact that multiple patterns co-exist in evolutionary data that, together, give rise to emergent protein functions

Model Code from Errors in mutagenesis and the benefit of cell-to-cell signalling in the evolution of stress-induced mutagenesis

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Negative Epistasis and Evolvability in TEM-1 ß-Lactamase- The Thin Line between an Enzyme's Conformational Freedom and Disorder

Epistasis is a key factor in evolution since it determines which combinations of mutations provide adaptive solutions and which mutational pathways toward these solutions are accessible by natural selection. There is growing evidence for the pervasiveness of sign epistasis—a complete reversion of mutational effects, particularly in protein evolution—yet its molecular basis remains poorly understood. We describe the structural basis of sign epistasis between G238S and R164S, two adaptive mutations in TEM-1 ß-lactamase— an enzyme that endows antibiotics resistance. Separated by 10 Å, these mutations initiate two separate trajectories toward increased hydrolysis rates and resistance toward second and third-generation cephalosporins antibiotics. Both mutations allow the enzyme's active site to adopt alternative conformations and accommodate the new antibiotics. By solving the corresponding set of crystal structures, we found that R164S causes local disorder whereas G238S induces discrete conformations. When combined, the mutations in 238 and 164 induce local disorder whereby nonproductive conformations that perturb the enzyme's catalytic preorganization dominate. Specifically, Asn170 that coordinates the deacylating water molecule is misaligned, in both the free form and the inhibitor-bound double mutant. This local disorder is not restored by stabilizing global suppressor mutations and thus leads to an evolutionary cul-de-sac. Conformational dynamism therefore underlines the reshaping potential of protein's structures and functions but also limits protein evolvability because of the fragility of the interactions networks that maintain protein structure

Negative Epistasis and Evolvability in TEM-1 \u3b2-Lactamase\u2014The Thin Line between an Enzyme's Conformational Freedom and Disorder

Epistasis is a key factor in evolution since it determines which combinations of mutations provide adaptive solutions and which mutational pathways toward these solutions are accessible by natural selection. There is growing evidence for the pervasiveness of sign epistasis\u2014a complete reversion of mutational effects, particularly in protein evolution\u2014yet its molecular basis remains poorly understood. We describe the structural basis of sign epistasis between G238S and R164S, two adaptive mutations in TEM-1 \u3b2-lactamase\u2014 an enzyme that endows antibiotics resistance. Separated by 10 \uc5, these mutations initiate two separate trajectories toward increased hydrolysis rates and resistance toward second and third-generation cephalosporins antibiotics. Both mutations allow the enzyme's active site to adopt alternative conformations and accommodate the new antibiotics. By solving the corresponding set of crystal structures, we found that R164S causes local disorder whereas G238S induces discrete conformations. When combined, the mutations in 238 and 164 induce local disorder whereby nonproductive conformations that perturb the enzyme's catalytic preorganization dominate. Specifically, Asn170 that coordinates the deacylating water molecule is misaligned, in both the free form and the inhibitor-bound double mutant. This local disorder is not restored by stabilizing global suppressor mutations and thus leads to an evolutionary cul-de-sac. Conformational dynamism therefore underlines the reshaping potential of protein's structures and functions but also limits protein evolvability because of the fragility of the interactions networks that maintain protein structures