Structure of the Vif-binding domain of the antiviral enzyme APOBEC3G

Abstract

The human APOBEC3G (A3G) DNA cytosine deaminase restricts and hypermutates DNA-based parasites including HIV-1. The viral infectivity factor (Vif) prevents restriction by triggering A3G degradation. While the structure of the A3G catalytic domain is known, the structure of the N-terminal Vif-binding domain has proven more elusive. Here, evolution- and structure-guided mutagenesis was used to solubilize the Vif-binding domain of A3G permitting structural determination by NMR spectroscopy. A smaller zinc-coordinating pocket and altered helical packing distinguish it from catalytic domain structures, and help explain the reported inactivity of this domain. This soluble A3G N-terminal domain is bound by Vif, which enabled mutagenesis and biochemical experiments to identify a unique Vif-interacting surface formed by α1-β1, β2-α2, and β4-α4 loops. This structure sheds new light on the Vif-A3G interaction and provides critical information for future drug development.

1.92 Angstrom Zinc-Free APOBEC3F Catalytic Domain Crystal Structure

“…The alpha 2 helix has a His-X-Glu motif and the alpha 3 helix has a Cys-Pro-X 2–4 -Cys motif that together coordinate a single zinc ion. This is best evidenced by crystal and solution structures of A3A [9, 10], A3Bctd [11], A3C [12], A3Fctd ([13–15] and this study), A3Gntd [16], and A3Gctd [17–22]. Importantly, prior to the current work, all crystal structures have contained a single zinc ion coordinated by the His-X-Glu and Cys-Pro-X 2–4 -Cys motifs.…”

The APOBEC Protein Family: United by Structure, Divergent in Function

“…The first APOBEC crystal structure obtained was for A2 with a 40-residue N-terminal deletion [95]. Subsequently, NMR solution structures of full-length A2 [96], the A3G C-terminal half [97–99], A3A [100], and the A3G N-terminal half [101], were reported as well as X-ray structures of A3C [102], the A3G C-terminal half [103–105], the A3B C-terminal half [106], and the A3F C-terminal half [107–109]. A low-resolution small-angle X-ray scattering model of recombinant full-length A3G also has been reported [110].…”

“…( A ) Cartoon representation of the solution NMR structures of A2Δ40 (PDB 2RPZ) [96], A3A (PDB 2M65) [100], and A3G (N-terminal half, PDB 2MZZ [101]) and X-ray crystal structures of A3B (C-terminal half, PDB 5CQI [106]), A3C (PDB 3VOW) [102], A3F (C-terminal half, PDB 4J4J [108]) and A3G (C-terminal half, PDB 3IR2 [104]) are depicted in the same orientation as that shown in [Text Box 4]. A3 Z1-type CDA domains are green and Z2-types are orange.…”

Redoxal, an inhibitor of de novo pyrimidine biosynthesis, augments APOBEC3G antiviral activity against human immunodeficiency virus type 1

“…1A) (Mehle et al, 2007; Pery et al, 2009; Pery et al, 2015). In this assay, interaction between a purified GST-Vif protein fragment that includes the A3G binding site (1–94 amino acids; GST-Vif) (Dang et al, 2010a; Dang et al, 2010b; Kouno et al, 2015; Mehle et al, 2007; Pery et al, 2009; Russell and Pathak, 2007; Yamashita et al, 2008), and a synthetic biotinylated A3G peptide corresponding to amino acids 110–148, which encompasses the Vif-binding site (Bogerd et al, 2004; Huthoff and Malim, 2007; Mangeat et al, 2004; Schrofelbauer et al, 2004), is detected by Europium (Eu-donor fluorophore)-labeled anti-GST antibodies and Streptavidin-Ulight (acceptor fluorophore). Interaction between Vif and A3G brings Eu and Ulight into close proximity, supporting energy transfer between these molecules; this energy transfer is then measured as a FRET signal and attenuation of Vif-A3G interaction results in signal reduction.…”