Compensatory evolution within proteins : An experimental assessment

Amino acids within a protein sequence interact to maintain their structure and function. Consequently, mutations at a given functionally significant position can be potentially compensated by a mutation at an interacting position. For instance, a substitution from an amino acid with a small side chain to an amino-acid with a big side chain can be compensated by a reciprocal substitution at an interacting position. Such a coevolution scenario implies that the first mutation leads to a fitness reduction, while the compensating mutation restores it. Several methods have been developed to detect coevolving positions from sequence alignments. However, the validation of the resulting predictions relies so far only on indirect evidence such as residue contact maps in proteins for which an experimental structure is available. This study used the dataset that mapped substitutions for a protein sequence alignment on each phylogeny branch to detect coevolving amino-acids in bacterial homologous protein families (CoMap). Accounting for the biochemical properties of amino acids, we short-listed potential candidates from a dataset of thousands of coevolving groups for the experimental assessment. We then selected three candidate groups from three proteins (Elongation factor 4, IspH, and YebC proteins) displaying a pattern of co-substitutions in the \textit{Escherichia coli} branch of the phylogeny for charge and beta propensities for compensation. I experimentally reconstructed the local fitness landscape, resurrecting the ancestral genotype in \textit{E. coli} and putting it in competition with single mutants and reconstructed ancestral state (double mutant). I have performed competition experiments in LB Broth nutrient-enriched medium and M9 minimal (glucose as a single carbon source). In EF4 and YebC proteins, I observed a valley of lower fitness in single mutants in the local fitness landscape and restoration peak in the ancestral state. I report the first experimental assessment of the prediction of coevolution within a protein. I have also observed a peak in one of the single mutants of IspH candidates, which compensates for the charge only in one direction of the mutation. We used direct measurement of fitness estimation in \textit{E. coli} rather than using a proxy for fitness, \textit{i.e.}, protein fitness mentioned in most studies. The results of this study provide experimental evidence of the compensating nature in two out of three tested candidates, highlighting the potential of coevolution detection methods as tools to understand molecular evolution. This study helps to understand the interaction of the amino acids in the 3D space and can be used to predict the mechanism of evolution.


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