Statistical coevolution analysis and molecular dynamics: Identification of amino acid pairs essential for catalysis

Abstract

Molecular dynamics (MD) simulations of HhaI DNA methyltransferase and statistical coupling analysis (SCA) data on the DNA cytosine methyltransferase family were combined to identify residues that are coupled by coevolution and motion. The highest ranking correlated pairs from the data matrix product (SCA⅐MD) are colocalized and form stabilizing interactions; the anticorrelated pairs are separated on average by 30 Å and form a clear focal point centered near the active site. We suggest that these distal anticorrelated pairs are involved in mediating active-site compressions that may be important for catalysis. Mutants that disrupt the implicated interactions support the validity of our combined SCA⅐MD approach.anticorrelated motion ͉ correlated motion ͉ M.HhaI ͉ statistical coupling analysis T he proposal that protein dynamics contributes significantly to enzyme catalysis is intriguing (1-4) yet is supported by limited experimental evidence. Previous studies have shown that correlated and anticorrelated motions within an enzyme's active site enhance the reaction rate by various mechanisms that increase the relative amounts of reactive orientations (5). These active-site fluctuations are proposed to result from motions involving distal structural elements and interconnecting networks (1-4). This hypothesis is indirectly supported by emerging molecular dynamics (MD) (1, 2), NMR (6, 7), and hybrid approaches (8-12). The MD studies, although difficult to verify experimentally, have provided highly suggestive results relating dynamics to catalysis. Ultimately, the quantitative contribution to catalysis of various dynamic mechanisms requires direct experimental testing. We combined MD simulations and a coevolution analysis [statistical coupling analysis (SCA); ref. 13] to identify residues that are coupled by coevolution and motion.Although MD simulations reveal active-site correlated and anticorrelated motions, the identity and role of specific structural elements outside the active site in mediating such motions is difficult to assign. For example, MD cross-correlation analyses are dominated by anticorrelated motions occurring between the most distal regions of protein, often residing in distinct domains (5). Although MD simulations implicate regions of allowed motion, the identity of single amino acids that facilitate these motions is not forthcoming and hence difficult for protein engineers to test. SCA identifies the functional coupling of specific residue pairs that in many cases are distal in the three-dimensional structure. The coupling of such residues leads to their coevolution and is revealed by the statistical analysis of hundreds of related sequences; this approach recently was validated by NMR and protein engineering studies (13-16). These applications of SCA have been focused on protein-ligand interactions, and here we apply SCA toward protein dynamics and catalysis.M.HhaI is one of many S-adenosylmethionine (AdoMet)-dependent DNA-modifying enzymes found in bacteria, plants, and animals (17). These enzym...

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Evolutionary Basis for the Coupled-domain Motions in Thermus thermophilus Leucyl-tRNA Synthetase

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