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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. Although the structure of the A3G catalytic domain is known, the structure of the N-terminal Vif-binding domain has proven more elusive. Here, we used evolution- and structure-guided mutagenesis to solubilize the Vif-binding domain of A3G, thus permitting structural determination by NMR spectroscopy. A smaller zinc-coordinating pocket and altered helical packing distinguish the structure from previous catalytic-domain structures and help to explain the reported inactivity of this domain. This soluble A3G N-terminal domain is bound by Vif; this enabled mutagenesis and biochemical experiments, which identified a unique Vif-interacting surface formed by the 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.

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Figure 1: Generation of soluble N-terminal Vif-binding domain of A3G.
Figure 2: Solution structure of the Vif-binding domain of A3G.
Figure 3: Structure comparison of sNTD with other APOBEC3 proteins.
Figure 4: Functional assays of sNTD.
Figure 5: Identification of Vif-binding regions of sNTD.

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Acknowledgements

This work was supported by grants from the US National Institutes of Health (AI073167 to H.M. and GM091743 to R.S.H.). Salary support for T.K. was provided in part by a Toyobo Biotechnology Foundation Fellowship. The University of Minnesota Supercomputing and NMR center (US National Science Foundation, BIR-961477) provided NMR instrumentation. We thank C. Kojima (Institute for Protein Research, Osaka University) for the pCold vector, K. Strebel (US National Institutes of Health) for the pcDNA-hVif plasmid, M. Katahira for advice on structure calculations, Y. Iwatani for advice on degradation assays, Y. Xia for advice on NMR experiments, Y. Hong for supporting preparation of expression vectors, F. Liu for advice on human-cell experiments and K. Walters for editing the manuscript.

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T.K. designed the strategy for searching for soluble A3G NTD mutants and all A3G NTD mutants; performed harvest assays, Vif binding assays in E. coli, Vif-dependent degradation assays and deamination assays; prepared NMR samples; and determined the solution structure. E.M.L. performed single-cycle infectivity assays. M.S. optimized human-cell experiments. S.M.D.S. analyzed surface-charge distribution and binding-pocket volumes of the NTD structure. J.Z. provided support in harvest assays. L.C. and M.H. provided support with preparation of expression plasmids and with the Vif binding assay in E. coli. T.K., E.M.L., S.M.D.S., C.A.S., R.S.H. and H.M. contributed to writing and editing the manuscript.

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Correspondence to Hiroshi Matsuo.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Amino acid substitutions for increasing solubility of the A3G N-terminal domain.

(a) Amino acid sequence alignments of A3G NTD (NP_068594), sNTD (this study), consensus (this study), A3G CTD (NP_068594), A3B CTD (NP_004891) and A3A (NP_663745). Identical and mismatched amino acid residues between wild-type NTD and sNTD are shown in red and gray, respectively. Residue numbers correspond to wild-type A3G NTD. (b) Schematic presentation of amino acid sequences of sNTD and its variants. Residues identical to wild-type NTD are illustrated in red, whereas those that differ are indicated in gray. Black triangles indicate the location of residues that were substituted back to their endogenous amino acid identity with sNTD as a template. (c) Results of protein harvest assays showing solubility of sNTD variants. The band intensities of GST fused sNTD (lane 1), wild-type A3G NTD (lane 2), A3G CTD-2K3A (lane 3), and sNTD variants, including #1 - #12 of figure 1b, in a Coomassie gel were analyzed and normalized using the value of sNTD. The mean values ± s. d. for n = 3 independent cell cultures is plotted.

Supplementary Figure 2 Cα region of 1H, 13C HSQC spectra acquired on sNTD samples with selective 1H- and 13C-labeling by amino acid type.

Spectrum acquired on sNTD that was (a) uniformly 13C-labeled, (b) [1H, 13C]Ala-labeled, (c) [1H, 13C]Phe-labeled, (d) [1H, 13C]Ile-labeled, (e) [1H, 13C]Lys-labeled, (f) [1H, 13C]Leu-labeled, (g) [1H, 13C]Met-labeled, (h) [1H, 13C]Arg-labeled, (i) [1H, 13C]Thr-labeled, (j) [1H, 13C]Tyr-labeled, and (k) [1H, 13C]Val-labeled. Assignments are provided in spectra.

Supplementary Figure 3 1H-15N TROSY spectrum of sNTD.

Assignments of backbone amide protons are presented next to signals. Signals labeled with asterisks seem to arise from minor conformations. The inlet figure shows that major and minor signals for E38, V39 and K40.

Supplementary Figure 4 β-sheet conformation found in sNTD.

Inter-strand NOE connectivities are shown by double-headed arrows, and hydrogen bonds identified using an H/D-exchange experiment are indicated with red oval rectangles.

Supplementary Figure 5 Deamination and Vif binding assays.

(a-f) Deamination assays. 10mer DNA fragment, d(5’-ATTCCCAATT-3’), was used as a substrate for the deamination reaction. (a-c) display spectra of the substrate (a), the first deamination product d(5’-ATTCCUAATT-3’) (b) and the second deamination product d(5’-ATTCUUAATT-3’) (c). (d-e) Spectra of the substrate incubated with A3G CTD-2K3A for 30 minutes (d) and 24 hours (e). (f) Spectrum of the substrate after incubation with sNTD for 24 hours. (g) Representative SDS-PAGE gel images of the E. coli co-expression and GST-pull down assays used to identify residues critical for sNTD binding to Vif. Substituted residues are indicated on top of each gel image. In each gel, P, S and B show the amount of protein in pellet, soluble fraction and GST resin, respectively. The position of each protein is shown on the right side of the figure.

Supplementary Figure 6 Structural features of the zinc-coordinating region of sNTD.

(a) Stereoview of the zinc coordinating region of sNTD. α2 and α3 helices are colored green, the zinc ion is colored purple, and side chain atoms of zinc-coordinating residues, including H65, C97 and C100, and E67, are shown in ball-and-stick representation. Side chain atoms of L35, W90, I92 and M104 are located close to the zinc ion (see main text). (b) Comparison of the substrate-binding pocket and surface properties of sNTD, the A3G-NTD homology model (A3G NTD), and A3G-CTD. Top panels: Substrate-binding pocket volumes are presented as orange blobs, with the protein molecular surface shown as dark-blue mesh. Bottom panels: Molecular surface electrostatic potential are shown in red and blue for negative and positive charges, respectively. (bottom). Orientation of molecule is shown on left by cartoon models (Zn2+ is shown in purple). Location of D128 residue is shown in NTD structures. (c-d) A hydrophobic core formed by β1-α2-β3-α3. (c) Amino acid sequence alignment of wild-type A3G NTD, sNTD and A3 domains with available structures. Residue numbers refer to A3G NTD. Residues involved in the hydrophobic core are colored red. (d) A stereoview of the hydrophobic core in the sNTD structure.

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Supplementary Data Set 1

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Kouno, T., Luengas, E., Shigematsu, M. et al. Structure of the Vif-binding domain of the antiviral enzyme APOBEC3G. Nat Struct Mol Biol 22, 485–491 (2015). https://doi.org/10.1038/nsmb.3033

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