Structural Mechanism of Substrate Specificity in Human Cytidine Deaminase Family APOBEC3s

APOBEC3s proteins (A3s), a family of cytidine deaminases, protect the host cell from endogenous retro‐elements and exogenous viral infections by introducing hypermutations. However, ability to mutate genomic DNA makes A3s a potential cancer source, as has been reported for A3A, A3B and A3H. Despite overall similarity, A3 proteins have distinct deamination activity and substrate specificity. Over the past years, several crystal and NMR structures of apo A3s and DNA/RNA‐bound A3s have been determined. These structures have suggested the importance of the loops around the active site for nucleotide specificity and binding. However, the structural mechanism underlying A3 activity and substrate specificity requires further examination. Using advanced molecular modeling followed by experimental mutational analysis and molecular dynamics (MD) simulations, we revealed the structural basis for DNA binding and relatively lower catalytic activity of A3B compared to the highly similar A3A. We identified Arg211 in loop 1 as the gatekeeper coordinating DNA, and critical residues for nucleotide specificity. We also identified a unique auto‐inhibited conformation in A3B that restricts access to the active site and may underlie lower catalytic activity. Recently, we investigated the structural mechanism for exclusion of RNA from A3G catalytic activity using similar methods. Overall, the comprehensive structural analysis of A3 domains in this project will reveal determinants of substrate specificity and shed light into the biological function of these enzymes, potentially leading to discover inhibitors to benefit cancer therapeutics. In addition, rational engineering of A3 domains to achieve varying catalytic efficiencies or distinct substrate specificity will be greatly beneficial for CPRISR–A3 base editing systems in gene therapy. Support or Funding Information This work is supported by the U.S. National Institutes of Health Institute of General Medical Sciences [R01GM118474].