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

Department of Biochemistry, Molecular Biology and Biophysics, [of Minnesota, Minneapolis, Minnesota 55455, USA.
Nature (Impact Factor: 41.46). 04/2008; 452(7183):116-9. DOI: 10.1038/nature06638
Source: PubMed


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.

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Available from: Keisuke Shindo, Dec 27, 2013
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    • "These mutational studies led us to examine the role of this methionine in A3B structure and whether it is conserved in the AID/APOBEC family of proteins. We first aligned A3B CTD sequence with the sequences of these enzymes using Clustal Omega software [36] and manually adjusted it to conserve secondary structure elements found within 3D (3-dimensional) structures of CTDs of two doubledomain APOBEC3 proteins (A3F and A3G) and complete structures of two single-domain APOBEC3 proteins (A3A and A3C) [18] [19] [23] [24] [37] [38]. The alignment suggests that Met193 is conserved among these cytosine deaminases (Fig. 7). "
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    ABSTRACT: Human APOBEC3B deaminates cytosines in DNA and belongs to the AID/APOBEC family of enzymes. These proteins are involved in innate and adaptive immunity, and may cause mutations in a variety of cancers. To characterize its ability to convert cytosines to uracils, we tested several derivatives of APOBEC3B gene for their ability to cause mutations in Escherichia coli. Through this analysis, a methionine residue at the junction of amino- and carboxy-terminal domains was found to be essential for high mutagenicity. Properties of mutants with substitutions at this position, examination of existing molecular structures of APOBEC3 family members and molecular modeling suggest that this residue is essential for the structural stability of this family of proteins. The APOBEC3B carboxy-terminal domain (CTD) with the highest mutational activity was purified to homogeneity and its kinetic parameters were determined. Size exclusion chromatography of the CTD monomer showed that it is in equilibrium with its dimeric form and MALDI-TOF analysis of the protein suggested that the dimer may be quite stable. The partially purified amino-terminal domain did not show intrinsic deamination activity and did not enhance the activity of the CTD in biochemical assays. Finally, APOBEC3B was at least 10-fold less efficient at mutating 5-methylcytosine (5mC) to thymine than APOBEC3A in a genetic assay, and was at least 10-fold less efficient at deaminating 5mC compared to C in biochemical assays. These results shed light on the structural organization of APOBEC3B catalytic domain, its substrate specificity and its possible role in causing genome-wide mutations. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Aug 2015 · Journal of Molecular Biology
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    • "Both distributions are rather narrow with maxima corresponding to the monomeric state of each protein. This finding is in line with the NMR results for A3A [20] and A3Gctd [36], [37] and the crystal structures of A3Gctd [36], [38], [39]. "
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    ABSTRACT: The APOBEC3 family of DNA cytosine deaminases functions to block the spread of endogenous retroelements and retroviruses including HIV-1. Potency varies among family members depending on the type of parasitic substrate. APOBEC3A (A3A) is unique among the human enzymes in that it is expressed predominantly in myeloid lineage cell types, it is strongly induced by innate immune agonists such as type 1 interferon, and it has the capacity to accommodate both normal and 5-methyl cytosine nucleobases. Here we apply atomic force microscopy (AFM) to characterize the interaction between A3A and single- and double-stranded DNA using a hybrid DNA approach in which a single-stranded region is flanked by defined length duplexes. AFM image analyses reveal A3A binding to single-stranded DNA, and that this interaction becomes most evident (∼80% complex yield) at high protein-to-DNA ratios (at least 100∶1). A3A is predominantly monomeric when bound to single-stranded DNA, and it is also monomeric in solution at concentrations as high as 50 nM. These properties agree well with recent, biochemical, biophysical, and structural studies. However, these characteristics contrast with those of the related enzyme APOBEC3G, which in similar assays can exist as a monomer but tends to form oligomers in a concentration-dependent manner. These AFM data indicate that A3A has intrinsic biophysical differences that distinguish it from APOBEC3G. The potential relationships between these properties and biological functions in innate immunity are discussed.
    Full-text · Article · Jun 2014 · PLoS ONE
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    • "The crystal structure of A3G shows a shallow groove in A3G that surrounds the catalytic core and is postulated to be the binding site for ssDNA [40]. A potential DNA-binding groove is also apparent in the NMR structure. "
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    ABSTRACT: APOBEC3A (A3A), one of the seven-member APOBEC3 family of cytidine deaminases, lacks strong antiviral activity against lentiviruses but is a potent inhibitor of adeno-associated virus and endogenous retroelements. In this report, we characterize the biochemical properties of mammalian cell-produced and catalytically active E. coli-produced A3A. The enzyme binds to single-stranded DNA with a Kd of 150 nM and forms dimeric and monomeric fractions. A3A, unlike APOBEC3G (A3G), deaminates DNA substrates nonprocessively. Using a panel of oligonucleotides that contained all possible trinucleotide contexts, we identified the preferred target sequence as TC (A/G). Based on a three-dimensional model of A3A, we identified a putative binding groove that contains residues with the potential to bind substrate DNA and to influence target sequence specificity. Taking advantage of the sequence similarity to the catalytic domain of A3G, we generated A3A/A3G chimeric proteins and analyzed their target site preference. We identified a recognition loop that altered A3A sequence specificity, broadening its target sequence preference. Mutation of amino acids in the predicted DNA binding groove prevented substrate binding, confirming the role of this groove in substrate binding. These findings shed light on how APOBEC3 proteins bind their substrate and determine which sites to deaminate.
    Full-text · Article · May 2014 · PLoS ONE
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