Fig 1.
Biochemical analysis of Vprmus-induced CRL4DCAF1 specificity redirection.
(A-C) GF analysis of in vitro reconstitution of protein complexes containing DDB1/DCAF1-CtD, Vprmus and SAMHD1 (A), SAMHD1-ΔCtD (B) or T4L-SAMHD1-CtD (C). Elution volumes of protein molecular weight standards are indicated above the chromatogram in A. Coomassie blue-stained SDS-PAGE analyses of fractions collected during the GF runs are shown below the chromatograms, with boxes colour-coded with respect to the chromatograms. SAM–sterile α-motif domain, HD–histidine-aspartate domain, T4L –T4 Lysozyme. The asterisk and double asterisk indicate slight contaminations with remaining GST-3C protease and the GST purification tag, respectively. (D-G) In vitro ubiquitylation reactions with purified protein components in the absence (D) or presence (E-G) of Vprmus, with the indicated SAMHD1 constructs as substrate. Reactions were stopped after the indicated times, separated on SDS-PAGE and visualised by Coomassie blue staining.
Fig 2.
Crystal structure of the DDB1/DCAF1-CtD/Vprmus complex.
(A) Overall structure of the DDB1/DCAF1-CtD/Vprmus complex in two views. DCAF1-CtD is shown as grey cartoon and semi-transparent surface. Vprmus is shown as a dark green cartoon with the co-ordinated zinc ion shown as grey sphere. T4L and DDB1 have been omitted for clarity. (B) Superposition of apo-DCAF1-CtD (light blue cartoon) with Vprmus-bound DCAF1-CtD (grey/green cartoon). Only DCAF1-CtD regions with significant structural differences between apo- and Vprmus-bound forms are shown. Disordered loops are indicated as dashed lines. (C) Comparison of the binary Vprmus/DCAF1-CtD and ternary Vpxsm/DCAF1-CtD/SAMHD1-CtD complexes. For DCAF1-CtD, only the N-terminal “acidic loop” region is shown. Vprmus, DCAF1-CtD and bound zinc are coloured as in A; Vpxsm is represented as orange cartoon and SAMHD1-CtD as pink cartoon. Selected Vpr/Vpx/DCAF1-CtD side chains are shown as sticks, and electrostatic interactions between these side chains are indicated as dotted lines. (D) GF analysis of in vitro reconstitution of protein complexes containing DDB1/DCAF1-CtD/Vprmus or the Vprmus R15E/R75E mutant, and SAMHD1. SDS-PAGE analyses of corresponding GF fractions are shown below the chromatogram, with boxes colour-coded with respect to the chromatogram. (E-F) In vitro reconstitution of protein complexes containing SAMHD1 and Vprmus R15E/R75E (E) or DDB1/DCAF1-CtD and Vprmus R15E/R75E (F). SDS-PAGE analyses of corresponding GF fractions are shown below the chromatogram, with boxes colour-coded with respect to the chromatogram. The asterisk and double asterisk indicate slight contaminations with remaining GST-3C protease and the GST purification tag, respectively.
Fig 3.
Cryo-EM analysis of CRL4-NEDD8DCAF1-CtD/Vprmus/SAMHD1 conformational states.
(A) Two views of an overlay of CRL4-NEDD8DCAF1-CtD/Vprmus/SAMHD1 cryo-EM reconstructions (conformational state-1 –light green, state-2 –salmon, state-3 –purple). The portions of the densities corresponding to DDB1 BPA/BPC, DCAF1-CtD and Vprmus have been superimposed. (B) Two views of a superposition of DDB1/DCAF1-CtD/Vprmus and CUL4/ROC1 (PDB 2hye) [15] molecular models, which have been fitted as rigid bodies to the corresponding cryo-EM densities; the models are oriented as in A. DDB1/DCAF1-CtD/Vprmus is shown as in Fig 2A, CUL4 is shown as cartoon, coloured as in A and ROC1 is shown as cyan cartoon. (C) Comparison of outermost CUL4 stalk orientations observed in the cryo-EM analysis presented here (states-1 and -3, coloured as in B, show 119.5° rotation of DDB1 BPB) to the two most extreme stalk positions present in previous crystal structures (PDB 4a0l [13], PDB 6dsz [123], coloured grey, show 143.4° DDB1 BPB rotation).
Fig 4.
Mechanism of SAMHD1-CtD recruitment by Vprmus.
(A) Two views of the cryo-EM reconstruction of the CRL4-NEDD8DCAF1-CtD/Vprmus/SAMHD1 core. The crystal structure of the DDB1/DCAF1-CtD/Vprmus complex was fitted as a rigid body into the cryo-EM density and is shown in the same colours as in Fig 2A. The DDB1 BPB model and density was removed for clarity. The red arrows mark additional density on the upper surface of the Vprmus helix bundle. (B) Detailed view of the SAMHD1-CtD electron density. The model is in the same orientation as in A, left panel. Selected Vprmus residues W29 and A66, which are in close contact to the additional density, are shown as red space-fill representation. (C) In vitro reconstitution of protein complexes containing DDB1/DCAF1-CtD, Vprmus or the Vprmus W29A/A66W mutant, and SAMHD1, assessed by analytical GF. SDS-PAGE analyses of corresponding GF fractions are shown below the chromatogram, with boxes colour-coded with respect to the chromatogram. (D-E) In vitro reconstitution of protein complexes containing SAMHD1 and Vprmus W29A/A66W (D) or DDB1/DCAF1-CtD and Vprmus W29A/A66W (E). SDS-PAGE analyses of corresponding GF fractions are shown below the chromatogram, with boxes colour-coded with respect to the chromatogram. The asterisk and double asterisk indicate slight contaminations with remaining GST-3C protease and the GST purification tag, respectively.
Fig 5.
Cross-linking mass spectrometry (CLMS) analysis of CRL4DCAF1-CtD/ Vprmus/SAMHD1.
(A) Schematic representation of sulfo-SDA cross-links between CRL4DCAF1/Vprmus and SAMHD1, identified by CLMS. Proteins are colour-coded as in Figs 3 and 4, and SAMHD1 black/white. SAMHD1-CtD is highlighted in yellow. Crosslinks to the N-terminal SAMHD1 globular SAM and HD domains are coloured light brown, while cross-links to the N-terminal half of SAMHD1-CtD are highlighted in pink and cross-links to the C-terminal end of SAMHD1-CtD are coloured purple. (B) Sulfo-SDA cross-links from A, in the same colour scheme, mapped on the molecular model of CRL4-NEDD8DCAF1-CtD/Vprmus/SAMHD1 (state-2), obtained from cryo-EM analysis (Fig 3). SAMHD1-CtD density from the CRL4-NEDD8DCAF1-CtD/Vprmus/SAMHD1 (core) cryo-EM analysis (Fig 4) is shown as yellow mesh. (C) The accessible interaction space of SAMHD1-CtD, calculated by the DisVis server [61], consistent with at least 14 of 26 observed cross-links, is visualised as grey mesh. DCAF1-CtD and Vprmus are oriented and coloured as in Fig 4A.
Fig 6.
Variability of neo-substrate recognition in Vpx/Vpr proteins.
Comparison of neo-substrate recognition modes of Vprmus (A), Vpxsm (B), Vpxmnd2 (C) and VprHIV-1 (D). DCAF1-CtD is shown as grey cartoon and semi-transparent surface, Vprmus−green, Vpxsm−orange, Vpxmnd2 –blue and VprHIV-1– light brown are shown as cartoon. Models of the recruited ubiquitylation substrates are shown as strongly filtered, semi-transparent calculated electron density maps with the following colouring scheme: SAMHD1-CtD bound to Vprmus−yellow, SAMHD1-CtD (bound to Vpxsm, PDB 4cc9) [50]–mint green, SAMHD1-NtD (Vpxmnd2, PDB 5aja) [51]–magenta, UNG2 (VprHIV-1, PDB 5jk7) [54]–light violet. (E) Multiple sequence alignment of Vpr and Vpx proteins from A-D. Helices are indicated by the boxes above the amino acid sequences. Residues involved in neo-substrate recognition are indicated by asterisks above the amino acid sequences. Residues involved in DCAF1-binding in all Vpr and Vpx proteins are indicated by red asterisks below the Vprmnd-2 amino acid sequence. Residues shaded grey or black are at least 60% or 90% type-conserved in all Vpx and Vpr proteins, respectively.
Fig 7.
Schematic illustration of structural plasticity in Vprmus-modified CRL4DCAF1-CtD, and implications for ubiquitin transfer.
(A) Rotation of the CRL4 stalk increases the space accessible to catalytic elements at the distal tip of the stalk, forming a ubiquitylation zone around the core. (B) Flexible tethering of SAMHD1 to the core by Vprmus places the bulk of SAMHD1 in the ubiquitylation zone and optimises surface accessibility. Under the experimental conditions, SAMHD1 adopts a monomer-dimer equilibrium, with both forms being competent for Vpr-binding. In B-C, only monomeric SAMHD1 is schematically indicated for clarity. (C) Modification of CUL4-WHB with NEDD8, triggered by substrate binding, leads to increased mobility of these distal stalk elements (CUL4-WHB, ROC1 RING domain) [56], further extending the ubiquitylation zone and activating the formation of a catalytic assembly for ubiquitin transfer (see also D) [72]. (D) Dynamic processes A-C together create numerous possibilities for assembly of the catalytic machinery (CUL4-NEDD8 WHB, ROC1, ubiquitin-(ubi-)charged E2) on surface-exposed SAMHD1 lysine side chains. Here, three of these possibilities are exemplified schematically. In this way, ubiquitin coverage on SAMHD1 is maximised.