Structure, interaction and real-time monitoring of the enzymatic reaction of wild-type APOBEC3G

Abstract

Human APOBEC3G exhibits anti-human immunodeficiency virus-1 (HIV-1) activity by deaminating cytidines of the minus strand of HIV-1. Here, we report a solution structure of the C-terminal deaminase domain of wild-type APOBEC3G. The interaction with DNA was examined. Many differences in the interaction were found between the wild type and recently studied mutant APOBEC3Gs. The position of the substrate cytidine, together with that of a DNA chain, in the complex, was deduced. Interestingly, the deamination reaction of APOBEC3G was successfully monitored using NMR signals in real time. Real-time monitoring has revealed that the third cytidine of the d(CCCA) segment is deaminated at an early stage and that then the second one is deaminated at a late stage, the first one not being deaminated at all. This indicates that the deamination is carried out in a strict 3 0 -5 0 order. Virus infectivity factor (Vif) of HIV-1 counteracts the anti-HIV-1 activity of APOBEC3G. The structure of the N-terminal domain of APOBEC3G, with which Vif interacts, was constructed with homology modelling. The structure implies the mechanism of species-specific sensitivity of APOBEC3G to Vif action.

HIV-1 Vif-mediated ubiquitination/degradation of APOBEC3G involves four critical lysine residues in its C-terminal domain

“…The structures of the individual domains are very similar to those of other cytidine deaminases and are characterized by a fivestranded mixed beta sheet and five alpha helices. The current CTD of our model is not substantially different from the CTD structures that were recently determined by X-ray crystallography and NMR (32,(37)(38)(39). Since we created and used our homology model to guide the current mutagenesis, several other models of A3G have become available (40,41).…”

A Structural Basis for the Biochemical Behavior of Activation-induced Deoxycytidine Deaminase Class-switch Recombination-defective Hyper-IgM-2 Mutants

“…4). A reduction in activity compared with WT AID begins to be apparent for ⌬C 17 , is much more pronounced for ⌬C 18 , and activity is abolished for ⌬C 19 and ⌬C 20 (Fig. 2, D and E).…”

“…Four other structures (three NMR and one crystal) of Apo3G-CD2 have also been solved (17)(18)(19)(20). Apo2 and Apo3G-CD2 show substantial overlap of an overall core structure containing a five ␤-strand sheet surrounded by six ␣-helices.…”

“…High resolution structures of the deaminase domain of APOBEC enzymes have been determined for Apo2 (16) and for the C-terminal catalytic domain (CD2) of Apo3G (17)(18)(19)(20). Overall, these structures showed a highly conserved deaminase domain with a five-stranded ␤-sheet core flanked by six ␣-helices (Fig.…”

Structural Model for Deoxycytidine Deamination Mechanisms of the HIV-1 Inactivation Enzyme APOBEC3G

“…S1). However, the CD2 domain does not contain the hydrophobic residues conserved in CD1 and Apo2; these residues have previously been shown to be involved in substrate specificity (29,36,37). The Apo3G C-mutant (23% monomer, 65% dimer, Fig.…”