Nuclear Magnetic Resonance Structure of the APOBEC3B Catalytic Domain: Structural Basis for Substrate Binding and DNA Deaminase Activity

Abstract

Human APOBEC3B (A3B) is a member of the APOBEC3 (A3) family of cytidine deaminases, which function as DNA mutators and restrict viral pathogens and endogenous retrotransposons. Recently, A3B was identified as a major source of genetic heterogeneity in several human cancers. Here, we determined the solution NMR structure of the catalytically active C-terminal domain (CTD) of A3B and performed detailed analyses of its deaminase activity. The core of the structure comprises a central five-stranded β-sheet with six surrounding helices, common to all A3 proteins. The structural fold is most similar to that of A3A and A3G-CTD, with the most prominent difference found in loop 1. The catalytic activity of A3B-CTD is ~15-fold less than that of A3A, although both exhibit similar pH dependence. Interestingly, A3B-CTD with an A3A loop 1 substitution had significantly increased deaminase activity, while a single residue change (H29R) in A3A loop 1 reduced A3A activity to the level seen with A3B-CTD. This establishes that loop 1 plays an important role in A3-catalyzed deamination by precisely positioning the deamination-targeted C into the active site. Overall, our data provide important insights into the determinants for the activities of individual A3 proteins and facilitate understanding of their biological function.

A Novel Regulator of Activation-Induced Cytidine Deaminase/APOBECs in Immunity and Cancer: Schrödinger’s CATalytic Pocket

“…This provided direct functional evidence that pocket closure limits activity. The second proof came from direct observation of closed pockets in several siblings of AID: in APOBEC3A by NMR (40), in APOBEC3B by X-ray crystallography (42, 57), and by NMR (43) (Figures 1C,D).…”

Determinants of Substrate Specificity in APOBEC3B

“…These positively charged loop 1 residues are known to be key for activity in A3A and A3Gctd, as A3A H29 and A3G H216 (homologous to A3B R212) could be mutated to an arginine while maintaining residual activity. 77,78 However, when A3G H216 is mutated to Ala, it loses activity. 78,79 While these backbone contacts appear to be charge driven and are not specific to one nucleotide sequence, the base atoms of the nucleotide make consistent hydrogen bonds with the loop1 backbone, in what may be a shape driven recognition process.…”

APOBEC3B lysine residues are dispensable for DNA cytosine deamination, HIV-1 restriction, and nuclear localization

“…1A). Recent crystal and solution structures show that the 2 lysines in the C-terminal domain are solvent-exposed and located near the catalytic site (Byeon et al, 2016; Shi et al, 2017; Shi et al, 2015) (Fig. 1B).…”