The APOBEC3B (A3B) single-stranded DNA (ssDNA) cytosine deaminase has important roles in innate immunity but is also a major endogenous source of mutations in cancer. Previous structural studies showed that the C-terminal catalytic domain of human A3B has a tightly closed active site, and rearrangement of the surrounding loops is required for binding to substrate ssDNA. Here we report structures of the A3B catalytic domain in a new crystal form that show alternative, yet still closed, conformations of active site loops. All-atom molecular dynamics simulations support the dynamic behavior of active site loops and recapitulate the distinct modes of interactions that maintain a closed active site. Replacing segments of A3B loop 1 to mimic the more potent cytoplasmic deaminase APOBEC3A leads to elevated ssDNA deaminase activity, likely by facilitating opening of the active site. These data collectively suggest that conformational equilibrium of the A3B active site loops, skewed toward being closed, controls enzymatic activity by regulating binding to ssDNA substrates.
Bibliographical noteFunding Information:
This work was supported by grants from the US National Institutes of Health (NIGMS R01-GM118000 to RSH and HA, NIGMS R35-GM118047 to HA, NCI R21-CA206309 to RSH, DP2-OD007237 and NIGMS P41-GM103426 to REA), the NSF (CHE060073N to REA), and the University of Minnesota Masonic Cancer Center (Spore-Program-Project-Planning Seed Grant to RSH). This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the US National Institutes of Health (NIGMS P41-GM103403). The Pilatus 6 M detector on 24-ID-C beamline is funded by a NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357, and those of the Minnesota Supercomputing Institute. R.S.H. is the Margaret Harvey Schering Land Grant Chair for Cancer Research, a Distinguished McKnight University Professor, and an Investigator of the Howard Hughes Medical Institute.