NMR structure of human restriction factor APOBEC3A reveals substrate binding and enzyme specificity

Abstract

Human APOBEC3A (A3A) is a single-stranded DNA (ssDNA) cytidine deaminase that restricts viral pathogens and endogenous retrotransposons and plays a role in the innate immune response. Furthermore, its potential to act as a genomic DNA mutator has implications for a role in carcinogenesis. A deeper understanding of A3A’s deaminase and nucleic acid binding properties, which is central to its biological activities, has been limited by the lack of structural information. Here, we report the NMR solution structure of A3A and show that the critical interface for interaction with ssDNA substrates includes residues extending beyond the catalytic center. Importantly, by monitoring deaminase activity in real time, we find that A3A displays similar catalytic activity on A3A-specific TTCA- or A3G-specific CCCA-containing substrates, involving key determinants immediately 5′ of the reactive C. Our results afford novel mechanistic insights into A3A-mediated deamination and provide the structural basis for further molecular studies.

Crystal Structure of the DNA Deaminase APOBEC3B Catalytic Domain

“…4, B and C). This improved activity relative to the A3Bctd-QM⌬loop3 parental construct is consistent with the more open active site conformation reported recently for A3A (34,40) (Fig. 3E).…”

“…The single dCMP molecule binds the A3Bctd surface and makes several contacts with the enzyme, but it is also assisted by crystal lattice contacts involving symmetry-related molecules (see PDB entry 5CQH), and thus the significance of this result should be interpreted conservatively. Nonetheless, the general location and orientation of the bound nucleotide appear consistent with previous models of A3 proteins binding ssDNA substrates (30,34,37,38,61,62). A3Gctd and A3A NMR chemical shift perturbation experiments, as well as mutational analyses of A3Fctd and A3Gctd, although differing in details, all suggest ssDNA binding paths spanning the catalytic domain surface and extending from the active site toward the ␣-helix 6, which overlaps with the crystallographically observed dCMP interacting with Arg-372 from the ␣-helix 6 and Tyr-319/Lys-320 from the N terminus of ␣-helix 4 (A3Bctd data in Fig.…”

“…As observed for the closely related Z1-type deaminase domains A3A and A3Gctd, ␤2 on the exposed edge of the ␤-sheet is interrupted by a short 3 10 helical segment (helix 1 in Fig. 2A; A3A (34,40); A3Gctd (30,31,33,37,39)). Correspondingly, the C-terminal end of ␣2, which interacts with this helical segment, is bent toward the discontinuous ␤2 (Fig.…”

“…Three of these substitutions (F200S, W228S, and L230K) changed A3Bctd residues to the corresponding sequence of the homologous A3A protein, which recently yielded to structural studies (34,40). F308K mimics the F302K and F310K substitutions used in structural studies of A3Fctd and A3Gctd, respectively (29 -31, 33).…”

A Biochemical Analysis Linking APOBEC3A to Disparate HIV-1 Restriction and Skin Cancer

“…A YYC motif preference is consistent with reported higher activities of Apo3A at physiological pH on short oligonucleotide substrates containing a pyrimidine at the 5Ј-side of C (42)(43)(44). Yet the analysis of motif specificity yields the surprising observation that there are roughly half as many mutations per clone at pH 5.5 compared with pH 8.0 (Fig.…”