Structure of the DNA deaminase domain of the HIV-1 restriction factor APOBEC3G

Abstract

The human APOBEC3G (apolipoprotein B messenger-RNA-editing enzyme, catalytic polypeptide-like 3G) protein is a single-strand DNA deaminase that inhibits the replication of human immunodeficiency virus-1 (HIV-1), other retroviruses and retrotransposons. APOBEC3G anti-viral activity is circumvented by most retroelements, such as through degradation by HIV-1 Vif. APOBEC3G is a member of a family of polynucleotide cytosine deaminases, several of which also target distinct physiological substrates. For instance, APOBEC1 edits APOB mRNA and AID deaminates antibody gene DNA. Although structures of other family members exist, none of these proteins has elicited polynucleotide cytosine deaminase or anti-viral activity. Here we report a solution structure of the human APOBEC3G catalytic domain. Five alpha-helices, including two that form the zinc-coordinating active site, are arranged over a hydrophobic platform consisting of five beta-strands. NMR DNA titration experiments, computational modelling, phylogenetic conservation and Escherichia coli-based activity assays combine to suggest a DNA-binding model in which a brim of positively charged residues positions the target cytosine for catalysis. The structure of the APOBEC3G catalytic domain will help us to understand functions of other family members and interactions that occur with pathogenic proteins such as HIV-1 Vif.

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).…”

Active nuclear import and cytoplasmic retention of activation-induced deaminase

“…Since protein shape may strongly influence the ability to diffuse 30 , we used APOBEC2 (A2) as control. Given the homology with A2 and APOBEC3G [31][32][33] , the predicted three-dimensional structure of AID safely allows us to postulate that the monomers of A2 (25.7 kDa) and AID (23.9 kDa) will have a similar general folding, and therefore shape ( Supplementary Fig. 1).…”

“…NLSs often overlap with nucleic acid binding domains 44 . Indeed, Arg24 and Arg112 might be involved in DNA binding by comparison with APOBEC3G 31 . However, three import-deficient but catalytically active AID-A2 chimeras (#5, 19-22 and 34-36) provide separation of function between active import and nucleic acid binding.…”

APOBEC proteins and intrinsic resistance to HIV-1 infection

“…With respect to the likely mechanism of catalysis, the histidine and two cysteine residues are thought to coordinate a zinc ion that enables the catalytic glutamic acid to deprotonate a water molecule and generate the zinc hydroxide nucleophile that is necessary for the deamination of the pyrimidine ring of cytidine (Betts et al 1994;. Two recent structural studies, one on APOBEC2 and one on the carboxyterminal CDA domain of A3G (the domain that is catalytically active), reveal that the core elements of the CDA domain comprise a platform of five b-strands that is flanked on either side by a-helices and connecting loops (Prochnow et al 2007;Chen et al 2008).…”