Highly Organized but Pliant Active-site of DNA Polymerase beta: Compensatory Interaction Mechanisms in Mutant Enzymes by Dynamics Simulations and Energy Analyses

Comparison of crystal structures of DNA polymerases in various liganded forms suggests that they undergo large subdomain motions upon binding substrates. To link these conformational transitions with kinetic results describing catalytic efficiency and fidelity, we investigate the role of key DNA polymerase beta active site residues on subdomain motion through dynamic simulations of five single-residue mutants: Arg283Ala (R283A), Tyr271Ala (Y271A), Asp276Val (D276V), Arg258Lys (R258K), and Arg258Ala (R258A). Subdomain motions are significantly affected in all mutant simulations. However, movement toward an active closed state was only observed in the R258A simulation, suggesting that Arg258 has a crucial role in modulating this motion that precedes chemistry. Analyses of protein/DNA interactions in the mutant active site also indicate distinctive hydrogen bonding and van der Waals patterns arising from compensatory structural adjustments. By comparing the closed substrate mutant complexes with the wild-type enzyme, we interpret experimentally derived nucleotide binding a finities in molecular terms: R283A (decreased), Y271A (slightly increased), D276V (increased), and R258A (decreased). Our findings demonstrate that although direct interactions with the incoming nucleotide may decrease (e.g.,Y271A), there are compensatory interactions (e.g., with adjacent residues Phe272, Asn279, and Arg283)that are predicted to increase the overall binding affinity for the incoming nucleotide. Taken together with energetic analyses, these findings lead to experimentally testable predictions regarding other mutants: R258G might increase the rate of nucleotide insertion and maintain enzyme fidelity as R258A; fD276L might increase the nucleotide binding affinity more than D276V; and R283A/K280A might decrease the nucleotide binding a finity and increase misinsertion more than R283A. Our combined observations regarding key roles of specific residues (e.g., Arg258) and compensatory interactions echo the dual nature of the DNA polymerase active site, namely versatility and speci ficity. The polymerase active site should accommodate various base pairs in an ever-changing DNA sequence context while preserving fidelity underscoring an organized but pliant active site essential to enzyme function.

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