Structural determinants of HIV-1 Vif susceptibility and DNA binding in APOBEC3F

Abstract

The human APOBEC3 family of DNA cytosine deaminases serves as a front-line intrinsic immune response to inhibit the replication of diverse retroviruses. APOBEC3F and APOBEC3G are the most potent factors against HIV-1. As a countermeasure, HIV-1 viral infectivity factor (Vif) targets APOBEC3s for proteasomal degradation. Here we report the crystal structure of the Vif-binding domain in APOBEC3F and a novel assay to assess Vif-APOBEC3 binding. Our results point to an amphipathic surface that is conserved in APOBEC3s as critical for Vif susceptibility in APOBEC3F. Electrostatic interactions likely mediate Vif binding. Moreover, structure-guided mutagenesis reveals a straight ssDNA-binding groove distinct from the Vif-binding site, and an 'aromatic switch' is proposed to explain DNA substrate specificities across the APOBEC3 family. This study opens new lines of inquiry that will further our understanding of APOBEC3-mediated retroviral restriction and provides an accurate template for structure-guided development of inhibitors targeting the APOBEC3-Vif axis.

Figure 1. APOBEC3 and characterization of A3Fc-CD2

(a) Schematic of the seven A3 intrinsic immune restriction factors (A3A, A3B, A3C, A3D, A3F, A3G and A3H). The three classes of DNA cytosine deaminase domains, Z1, Z2 and Z3 are colored pink, green, blue, respectively. The Z2-cytosine deaminase domains are further classified into three subgroups based on sequence similarity. A3F and A3G are the two most potent A3 proteins and exhibit disparate Vif binding sites (CD1 for A3G and CD2 for A3F). (b) SDS-polyacrylamide gel electrophoresis of the purified A3Fc-CD2 after final size exclusion chromatography. (c) Biolayer interferometry kinetic analysis of A3Fc-CD2 binding to ssDNA. Biotin-labeled ssDNA was coupled to streptavidin-coated biosensors and monitored for binding to purified A3Fc-CD2 at 0, 0.4, 1, 2 and 4 μM concentrations. The data was analyzed based on a 1:1 binding model using the BLItz Pro software, with the fitted curves shown as grey lines. The ssDNA sequence used in the assay is shown below the sensorgram, with the A3Fc-CD2 deamination site and target DNA cytosine underlined and double underlined, respectively. (d) Rif^R mutation profile of E. coli expressed A3Fc-CD2 and A3G-CD2. Histogram showing the percent mutation on specific rpoB nucleotide sequences for A3Fc-CD2 and A3G-CD2. Results are expressed as the percentage of total mutations from six independent experiments with at least 20 Rif^R colonies sequenced for both A3Fc-CD2 and A3G-CD2.

Figure 2. Crystal structure of A3Fc-CD2

(a) Ribbon diagram of A3Fc-CD2. The chain is colored in a rainbow gradient from red (N-terminus) to blue (C-terminus). The catalytic zinc atom is labeled and shown as a grey sphere. The inset box shows a zoomed view of the catalytic site. (b) Structural difference between A3Fc-CD2, A3G-CD2 and A3C. All APOBEC3 cytosine deaminase domains have 10 loops. The largest structural deviations reside in loops L1, L2, L3, L4 and L7, colored orange, green, red, blue and purple, respectively. (c) Multiple sequence alignment of A3F-CD2, A3C, A3D-CD2, A3G-CD1, A3G-CD2 and A3H. Numbering of the sequences and depiction of secondary structural elements are based on A3Fc-CD2, and is shown above the sequences. Strictly conserved residues are highlighted in red. Putative A3Fc-CD2 ssDNA-binding and Vif-binding residues are outlined in purple and blue boxes, respectively. Previously identified residues in the hydrophobic V-shaped groove formed by the α2 and α3 helices are outlined in yellow. Catalytic site residues are denoted with an asterisk above its sequence. The sequence alignment was produced using Clustal W^, and the alignment graphics were generated using the program ESPript.

Figure 3. Identification of the A3Fc-CD2 ssDNA-binding site

(a) Molecular surface of the putative A3Fc-CD2 ssDNA-binding site. Key residues proposed for DNA binding are shown in purple. Aromatic and positively charges residues line the wall of a straight groove. This DNA binding groove is consistent with Model #1 in panel (c). Note: Residue W209 belongs to the A3G solubilization linker attached at the N-terminus of A3Fc-CD2. The native residue in A3Fc-CD2 is a lysine, and this residue is in position to accommodate the negative charges fro the phosphate backbone of the ssDNA strand. (b) A3Fc-CD2 active site. Catalytic and ssDNA binding residues are shown in cyan and purple sticks, respectively. (c) Two models of A3G-CD2 ssDNA binding. Model #1 is proposed by Furukawa, et al. and Chen, et al. and is based on a NMR titration analysis of ssDNA^,. DNA-binding residues were found to line a straight DNA binding groove. Model #2 is proposed by Holden, et al. and displays the DNA binding residues along a kinked groove. Both A3G-CD2 molecular surfaces are shown in the same orientation as panel (a). (d) Nucleic acid-protein interaction ELISA assay. Alanine scanning mutagenesis of selected A3Fc-CD2 ssDNA-binding site residues. dsDNA was used as a negative control. Results are expressed as the mean relative absorbance (+ standard deviation of the population) of three replicates. (e) Rif^R mutation profile of E. coli expressed WT A3G-CD2 and A3G-CD2 ‘YYFW’. Histogram showing the percent total mutation on specific rpoB nucleotide sequences. Results are expressed as the percentage of total mutations from six independent experiments with at least 20 colonies sequenced.

Figure 4. A3Fc-CD2 HIV-1 Vif-binding site

(a) Biolayer interferometry kinetic analysis of A3Fc-CD2 binding to refolded full length HIV-1 Vif. Biotin-labeled HIV-1 Vif was coupled to streptavidin-coated biosensors and monitored for binding to purified A3Fc-CD2 at 0, 0.4, 1, 2 and 4 μM concentrations. The data were analyzed based on a 1:1 binding model, as only one A3F deamination motif is found on the ssDNA. The calculated fitted curves are shown as grey lines. (b) Putative Vif-binding residues. A number of aromatic and hydrophobic residues, colored in yellow, (L255, F258, L263, Y269 and F290) are buried at the A3Fc-CD2 core. Residues L263 and S264 (shown in orange) were identified in this study to be not important for Vif binding.

Figure 5. Surface electrostatic potential of APOBEC3 proteins

A negatively charged surface conserved with other Z2-cytosine deaminase domains is proposed to be important for Vif-binding (shown in dashed lines). Electrostatic potential mapped onto the molecular surface of A3C (PDB: 3VOW), and the homology models of A3D-CD2, A3G-CD1 and A3H. The proposed footprints of the A3 negative and hydrophobic patch involved in Vif binding are shown by the solid and dashed lines, respectively. A previously characterized ‘DPD’ motif involved in A3G Vif binding is displayed for the A3G-CD1 homology model. Red and blue colored regions denote negative and positive charges, respectively. Note: the ssDNA-binding site is at the top of the depicted A3 molecules and has no overlap with the hydrophobic or negatively charged Vif-binding site.