The HIV-1 accessory protein Vpr is required for efficient viral infection of macrophages and promotion of viral replication in T cells. Vpr's biological activities are closely linked to the interaction with human DCAF1, a cellular substrate receptor of the Cullin4–RING E3 ubiquitin ligase (CRL4) of the host ubiquitin–proteasome-mediated protein degradation pathway. The molecular details of how Vpr usurps the protein degradation pathway have not been delineated. Here we present the crystal structure of the DDB1–DCAF1–HIV-1–Vpr–uracil-DNA glycosylase (UNG2) complex. The structure reveals how Vpr engages with DCAF1, creating a binding interface for UNG2 recruitment in a manner distinct from the recruitment of SAMHD1 by Vpx proteins. Vpr and Vpx use similar N-terminal and helical regions to bind the substrate receptor, whereas different regions target the specific cellular substrates. Furthermore, Vpr uses molecular mimicry of DNA by a variable loop for specific recruitment of the UNG2 substrate.
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We thank J. Skowronski for carefully reading the manuscript and providing us with valuable critical comments and suggestions. We also acknowledge M. Becker and C. Ogata at GM/CA (Argonne National Laboratory), and I. Mathews, C. Smith and A. Gonzalez at the Stanford Synchrotron Radiation Lightsource (SSRL) for their support during data collection. We thank D. Lee for home-source X-ray technical support. This work was supported by NIH grant P50GM082251 (to A.M.G.). G.C. acknowledges support from BioXFEL-STC1231306. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.
The authors declare no competing financial interests.
Integrated supplementary information
(A) The final refined 2Fo-Fc electron density map, contoured at 1.4σ of Vpr, illustrating well-defined side chains.(B–D) Three views of the final refined 2Fo-Fc electron density map of the Vpr (red)-UNG2 (green) interacting region, contoured at 1.4σ. (B) Electron density for both Vpr and UNG2 residues. (C) Electron density for UNG2 residues. (D) Electron density for Vpr residues.(E) Two views of the dimeric arrangement of the complex in the ASU.(F) Schematic depiction of the protein constructs used for crystallization. The color scheme for the individual proteins is used throughout the manuscript.(G) Crystal contacts across the ASU.(H) Multi-angle light scattering of the DDB1-DCAF1 (blue), DDB1-DCAF1-Vpr (red) and a mixture of DDB1-DCAF1-Vpr with excess of UNG2 (green). All samples were injected into an analytical Superdex200 gel filtration column. Molecular masses of DDB1-DCAF1, DDB1-DCAF1-Vpr, DDB1-DCAF1-Vpr-UNG2 and UNG2 were estimated to be 168 kDa, 182 kDa, 206 kDa, and 32 kDa, respectively.
(A) Ribbon representation of the Vpr structure in the DDB1-DCAF1-Vpr-UNG2 complex. (B) Electrostatic surface representation of Vpr, illustrating pertinent structural features important for binding UNG2 (green) and DCAF1 (blue). Vpr bridges UNG2 and DCAF1 and no direct contacts between the latter two are present in the structure. The contact area of Vpr with UNG2 (insert loop) is negatively charged, mimicking the negative charge of the DNA phosphate backbone.(C) Stereo-view of the best-fit superposition of the UNG2-DNA (PDB 2OYT)42 complex (brown and purple worms, respectively) and the UNG2-Vpr (green and red worms respectively) portion of the DDB1-DCAF1-Vpr-UNG2 complex structures. DIL: DNA intercalating loop. Conformational changes in UNG2 in these structures involve mainly the DNA binding groove and the DIL.(D) Two expanded views of (C), illustrating details of the interaction between UNG2 and DNA (brown and purple ribbons, respectively) and UNG2 and Vpr, (green and red ribbons, respectively). The left panel illustrates the close match in distances between residues in helices α1 and α2 and the minor groove phosphate backbone of the DNA. The right panel depicts residues in helices α1 and α2 that mimic the curvature of the minor groove phosphate backbone of the DNA.(E-F) Two expanded views of the superposition of the Vpr insert loop and the phosphate backbone around the abasic sugar, illustrating Vpr’s structural mimicry.(G-H) Structure of UNG2 (green ribbon) complexed with Ugi (cyan ribbon)40.(I) UNG2 base excision and inhibition assay using a ssDNA substrate in the presence and absence of Vpr. The control reaction was carried out without UNG2. DDB1-DCAF1 alone is not able to inhibit UNG2 deamination of the ssDNA substrate, while increasing amounts of DDB1-DCAF1-Vpr added to the reaction inhibits UNG2 activity.
(A) View of DCAF1 (blue, ribbon) bridging DDB1 (orange, surface) and Vpr (red, surface). Residues on the DCAF1 loops form the binding interface for Vpr. Residues on DCAF1's helix-loop-helix motif and residues on DCAF1 loops on the opposite site of the Vpr binding site form the DDB1 binding interface.(B-C) View down onto the DCAF1 WD40 propeller (electrostatic surface representation), illustrating Vpr (red, ribbon) binding. The surface ofthe β-propeller is asymmetric; loops connecting the β-strands on one hemi-solenoid of the propeller create the cleft where Vpr binds.(D) Two views of the interaction between the DCAF1 loops (thick trace, blue; β-strands blue worm) and Vpr (thin trace, red). Pertinent side chain contacts are depicted in the right side panel (stick representation).(E) Ribbon representation of the interaction between DCAF1 (blue) and DDB1 (orange) in the current structure.(F) Superposition of DCAF1 (blue) and DDB2 (green) (PDB 3EI1)49 bound to DDB1 (orange).(G) Structure of the complex between DDB2 (green) and DDB1 (orange) (PDB 3EI1)49.(H) Detail of the interaction between the helix-loop-helix region of DCAF1 (blue, ribbon) and DDB1 (orange, ribbon). Pertinent side chain interactions are depicted in stick representation.(I) Pull-down and western blot. Samples are lysates from HEK293 cells, transiently transfected with FLAG-tagged DCAF1 and Myc-tagged DDB1.(J-K) Two views of the superposition of the DCAF1 (blue)-Vpr (red) portion of the DDB1-DCAF1-Vpr-UNG2 complex with DDB2 (green)-DNA (purple) portion of the DDB1-DDB2-DNA complex (PDB 3EI1)49.
(A-C) Ribbon representation of VprHIV-1 (red), VpxMND (blue) and VpxSM (gray). The hydrophobic cleft in the three-helix bundle of Vpr into which Leu72 and Val74 from UNG2 bind is depicted by a green and black surface. Side chains that fill this pocket in VpxMND include Trp45 and Tyr67 and in VpxSM Trp49 and Tyr71.(D) DCAF1-Vpx-SAMHD1 structure (PDB 4Z8L)27, illustrating H-bonds between the acidic loop residues of DCAF1 (1090-1095) and Tyr62, Tyr65 and Arg66 of VpxMND (depicted in blue ball and stick) that coordinate interactions with Arg14 from SAMHD1 (depicted in yellow ball and stick)26,27. In contrast, Ala55 and Ala59 of Vpr in the DDB1-DCAF1-Vpr-UNG2 structure, depicted in red, do not form hydrogen bonds with the acidic loop of DCAF1.(E) Detail of the interactions between the N-terminal tails of VprHIV-1 (red) and VpxMND (blue) and DCAF1 (grey) in the DDB1-DCAF1-Vpr-UNG2 and DCAF1-Vpx-SAMHD1 complex structures, illustrating how SAMHD1's (yellow) N-terminal tail influences the positioning of N-terminus of Vpx. DCAF1 is depicted in surface representation while the other structures are shown in ribbon representation.(F) Expanded view of the interaction between the insert loop of VpxMND (blue) and SAMHD1 (yellow) in ribbon representation (PDB 4Z8L)27.(G) Structure-based sequence alignment of VprHIV-1, VpxMND and VpxSM proteins. The positions of the three helices are illustrated by red, blue and silver bars. Zn2+ coordinating His and Cys residues in all proteins are shown in red. DCAF1 interacting residues and UNG2 (for Vpr) or SAMHD1 interacting residues (for Vpx) are shown in blue and green, respectively.
(A) Superposition of the BPA domain of DDB1 from the DDB1-DCAF1-Vpr-UNG2 structure (orange surface and worm representation) with the BPA domain of the DDB1-CUL4A-RBX1-S5V structure (PDB 2HYE56, grey surface and worm representation) and the BPA domain from the DDB1-CUL4A-Rbx1-DDB2-DNA structure (PDB 4A0L46, blue surface and worm representation). The BPA and BPC domains in all three structures adopt similar conformations, while the BPB domains are rotated with respect to each other46.(B) Alternative view of (A) illustrating the relative disposition of the BPB domains in all three structures. In the inset, the different directions adopted by the first β-strand of BPB, following the hinge residues (388-392, superimposed) connecting BPA and BPB is shown.(C-D) Two views of a CUL4A-RBX1-DDB1-DCAF1-Vpr-UNG2 model, based on the superposition depicted in (A) and (B), using PDB 2HYE56, gray; PDB 4A0L46, blue). UNG2 gets positioned close to the RBX1 structure, thus poised for ubiquitination.
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Wu, Y., Zhou, X., Barnes, C. et al. The DDB1–DCAF1–Vpr–UNG2 crystal structure reveals how HIV-1 Vpr steers human UNG2 toward destruction. Nat Struct Mol Biol 23, 933–940 (2016). https://doi.org/10.1038/nsmb.3284
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