Adenine base editors (ABEs) have been subjected to multiple rounds of mutagenesis with the goal of optimizing their function as efficient and precise genome editing agents. Despite an ever-expanding data set of ABE mutants and their corresponding DNA or RNA-editing activity, the molecular mechanisms defining these changes remain to be elucidated. In this study, we provide a systematic interpretation of the nature of these mutations using an entropy-based classification model that relies on evolutionary data from extant protein sequences. Using this model in conjunction with experimental analyses, we identify two previously reported mutations that form an epistatic pair in the RNA-editing functional landscape of ABEs. Molecular dynamics simulations reveal the atomistic details of how these two mutations affect substrate-binding and catalytic activity, via both individual and cooperative effects, hence providing insights into the mechanisms through which these two mutations are epistatically coupled.
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