Abstract
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.
Highlights
The APOBEC3 family of single-stranded DNA cytosine deaminases plays important roles in innate immunity against viruses and transposons
The first crystal structures of A3Bctd showed that it has the canonical zinc-dependent deaminase fold consisting of a central single layer of β-sheet surrounded by α-helices[15], similar to other APOBEC3 members structurally characterized to date
The hydrogen-bond analysis of molecular dynamics (MD) trajectories revealed dynamic behavior the 206PLV208 region of A3B with three persistent inter-residue hydrogen-bonds; between Leu[207] backbone and Asp[205] side chain, Val[208] backbone and Arg[311] side chain, and Val[208] backbone and Asp[205] backbone (3%). These results strongly suggest that the higher activity conferred by the A3A loop 1 is not solely dependent on the identities of the amino acids in direct contacts with DNA, and likely depends on factors that affect the positioning and accessibility of these residues
Summary
The APOBEC3 family of single-stranded DNA (ssDNA) cytosine deaminases plays important roles in innate immunity against viruses and transposons (reviewed by refs[1,2,3]). As loop 1 residues play critical roles in binding ssDNA, the higher deaminase activity of A3A is likely conferred by residues within this loop, including His[29] that makes dual phosphate contacts to clamp down the target deoxycytidine nucleotide in the active site[25,30] (Figs 1B and 2B)[25,30]. These structural insights are supported by biochemical data using wild-type and mutant enzymes[14,16,25,31,32]. The smaller length of loop 1 of A3A, containing three fewer amino acids than A3Bctd loop 1, may be important for higher activity due to permitting increased active site accessibility
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