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Structural insights into Cullin4-RING ubiquitin ligase remodelling by Vpr from simian immunodeficiency viruses

['Sofia Banchenko', 'Charité Universitätsmedizin Berlin', 'Corporate Member Of Freie Universität Berlin', 'Humboldt-Universität Zu Berlin', 'Institute Of Medical Physics', 'Biophysics', 'Berlin', 'Ferdinand Krupp', 'Christine Gotthold', 'Jörg Bürger']

Date: 2021-08

Viruses have evolved means to manipulate the host’s ubiquitin-proteasome system, in order to down-regulate antiviral host factors. The Vpx/Vpr family of lentiviral accessory proteins usurp the substrate receptor DCAF1 of host Cullin4-RING ligases (CRL4), a family of modular ubiquitin ligases involved in DNA replication, DNA repair and cell cycle regulation. CRL4 DCAF1 specificity modulation by Vpx and Vpr from certain simian immunodeficiency viruses (SIV) leads to recruitment, poly-ubiquitylation and subsequent proteasomal degradation of the host restriction factor SAMHD1, resulting in enhanced virus replication in differentiated cells. To unravel the mechanism of SIV Vpr-induced SAMHD1 ubiquitylation, we conducted integrative biochemical and structural analyses of the Vpr protein from SIVs infecting Cercopithecus cephus (SIV mus ). X-ray crystallography reveals commonalities between SIV mus Vpr and other members of the Vpx/Vpr family with regard to DCAF1 interaction, while cryo-electron microscopy and cross-linking mass spectrometry highlight a divergent molecular mechanism of SAMHD1 recruitment. In addition, these studies demonstrate how SIV mus Vpr exploits the dynamic architecture of the multi-subunit CRL4 DCAF1 assembly to optimise SAMHD1 ubiquitylation. Together, the present work provides detailed molecular insight into variability and species-specificity of the evolutionary arms race between host SAMHD1 restriction and lentiviral counteraction through Vpx/Vpr proteins.

Due to the limited size of virus genomes, virus replication critically relies on host cell components. In addition to the host cell’s energy metabolism and its DNA replication and protein synthesis apparatus, the protein degradation machinery is an attractive target for viral re-appropriation. Certain viral factors divert the specificity of host ubiquitin ligases to antiviral host factors, in order to mark them for destruction by the proteasome, to lift intracellular barriers to virus replication. Here, we present molecular details of how the simian immunodeficiency virus accessory protein Vpr interacts with a substrate receptor of host Cullin4-RING ubiquitin ligases, and how this interaction redirects the specificity of Cullin4-RING to the antiviral host factor SAMHD1. The studies uncover the mechanism of Vpr-induced SAMHD1 recruitment and subsequent ubiquitylation. Moreover, by comparison to related accessory proteins from other immunodeficiency virus species, we illustrate the surprising variability in the molecular strategies of SAMHD1 counteraction, which these viruses adopted during evolutionary adaptation to their hosts. Lastly, our work also provides deeper insight into the inner workings of the host’s Cullin4-RING ubiquitylation machinery.

Funding: This research was supported by the German Research Foundation (DFG) Emmy Noether Programme SCHW1851/1-1 (D.S.), the DFG project grant 329673113 (J.R.), the DFG cluster of excellence EXC 2008 - 390540038 - UniSysCat (L.S., J.R., C.M.T.S.), the DFG research training group GRK 2473 - 392923329 - Bioactive Peptides - Innovative Aspects of Synthesis and Biosynthesis (L.S.) https://www.dfg.de/ , by an European Molecular Biology Organization (EMBO) Advanced laboratory start-up grant aALTF-1650 (D.S.) https://www.embo.org/ , by Wellcome Trust grants 108014/Z/15/Z (I.A.T.) and 103139 (J.R.), by the Wellcome Centre for Cell Biology, which is supported by core funding from the Wellcome Trust 203149 (J.R.) https://wellcome.org/ , and by an iNEXT instrumentation grant 3825 (D.S.) http://www.inext-eu.org/ . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: The coordinates and structure factors for the crystal structures have been deposited at the Protein Data Bank (PDB, https://www.rcsb.org/ ) with the accession codes 6ZUE (DDB1/DCAF1-CtD) and 6ZX9 (DDB1/DCAF1-CtD/T4L-Vprmus 1-92) ( https://doi.org/10.2210/pdb6ZUE/pdb and https://doi.org/10.2210/pdb6ZX9/pdb ). Cryo-EM reconstructions have been deposited at the Electron Microscopy Data Bank (EMDB, https://www.ebi.ac.uk/pdbe/emdb/ ) with the accession codes EMD-10611 (core), EMD-10612 (conformational state-1), EMD-10613 (state-2) and EMD-10614 (state-3) ( https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-10611 and https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-10612 and https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-10613 and https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-10614 ). CLMS data have been deposited at the PRIDE database and can be accessed via the following link: https://www.ebi.ac.uk/pride/archive/projects/PXD020453 .

To uncover the molecular mechanisms of DCAF1- and SAMHD1-interaction of such a “hybrid” Vpr, we initiated integrative biochemical and structural analyses of the Vpr protein from an SIV infecting Cercopithecus cephus, Vpr mus . These studies reveal similarities and differences to Vpx and Vpr proteins from other lentivirus species and pinpoint the divergent molecular mechanism of Vpr mus -dependent SAMHD1 recruitment to CUL4/ROC1/DDB1/DCAF1 (CRL4 DCAF1 ). Furthermore, cryo-electron microscopic (cryo-EM) reconstructions of a Vpr mus -modified CRL4 DCAF1 protein complex allow for insights into the structural plasticity of the entire CRL4 ubiquitin ligase assembly, with implications for the ubiquitin transfer mechanism.

In the course of evolutionary adaptation to their primate hosts, due to selective pressure to evade SAMHD1 restriction, two groups of SIVs, SIV agm and SIV deb/mus/syk , branched off from a common ancestor containing a Vpr protein which was unable to interact with SAMHD1, and neo-functionalised Vpr to bind SAMHD1 and induce its degradation. Subsequently, through a gene duplication or a recombination event, SIV and HIV clades exemplified by SIV rcm and HIV-2 gained the Vpx protein which took over the SAMHD1-degradation functionality. These viruses additionally encode for a Vpr protein with similar characteristics to the ancestral Vpr [ 24 , 49 , 53 ]. Consequently, SIV agm and SIV deb/mus/syk evolved “hybrid” Vpr proteins that retain targeting of some host factors depleted by HIV-1-type Vpr [ 27 ], and additionally induce SAMHD1 degradation.

By contrast, Vpx, exhibits a much narrower substrate range. It has recently been reported to target stimulator of interferon genes (STING) and components of the human silencing hub (HUSH) complex for degradation, leading to inhibition of antiviral cGAS-STING-mediated signalling and reactivation of latent proviruses, respectively [ 34 – 36 ]. Importantly, Vpx also recruits the SAMHD1 restriction factor to DCAF1, in order to mark it for proteasomal destruction [ 37 , 38 ]. SAMHD1 is a deoxynucleotide triphosphate (dNTP) triphosphohydrolase that restricts retroviral replication in non-dividing cells by lowering the dNTP pool to levels that cannot sustain viral reverse transcription [ 39 – 46 ]. Retroviruses that express Vpx are able to alleviate SAMHD1 restriction, allowing for replication in differentiated myeloid lineage cells, resting T cells and memory T cells [ 38 , 47 , 48 ]. As a result of the constant evolutionary arms race between the host’s SAMHD1 restriction and its viral antagonist Vpx, the mechanism of Vpx-mediated SAMHD1 recruitment is highly virus species- and strain-specific: The Vpx clade represented by Vpx HIV-2 recognises the SAMHD1 C-terminal domain (CtD), while Vpx mnd2/rcm binds the SAMHD1 N-terminal domain (NtD) in a fundamentally different way [ 24 , 49 – 52 ].

Vpr HIV-1 is important for virus replication in vivo and in macrophage infection models [ 26 ]. Recent proteomic analyses revealed that DCAF1 specificity modulation by Vpr HIV-1 proteins results in down-regulation of hundreds of host proteins in a DCAF1- and proteasome-dependent manner [ 27 ], including the previously reported Vpr HIV-1 degradation targets UNG2 [ 28 ], HLTF [ 29 ], MUS81 [ 30 , 31 ], MCM10 [ 32 ] and TET2 [ 33 ]. This surprising promiscuity in degradation targets is also partially conserved in more distant clades exemplified by Vpr agm and Vpr mus [ 27 ]. However, Vpr pleiotropy, and the lack of easily accessible experimental models, have prevented a characterisation of how these degradation events precisely promote replication [ 26 ].

Viral factors also bind to and modify DCAF receptors in order to redirect them to antiviral substrates. Prime examples are the lentiviral accessory proteins Vpr and Vpx. All contemporary human and simian immunodeficiency viruses (HIV/SIV) encode Vpr, while only two lineages, represented by HIV-2 and SIV infecting mandrills, carry Vpx [ 24 ]. Vpr and Vpx proteins are packaged into progeny virions and released into the host cell upon infection, where they bind to DCAF1 [ 25 ]. In this work, corresponding simian immunodeficiency virus Vpx/Vpr proteins will be indicated with their host species as subscript, with the following abbreviations used: mus–moustached monkey (Cercopithecus cephus), mnd–mandrill (Mandrillus sphinx), rcm–red-capped mangabey (Cercocebus torquatus), sm–sooty mangabey (Cercocebus atys), deb–De Brazza’s monkey (Cercopithecus neglectus), syk–Syke’s monkey (Cercopithecus albogularis), agm–african green monkey (Chlorocebus spec).

Frequently, virally encoded modifying proteins associate with, and adapt the Cullin4-RING ubiquitin ligases (CRL4) [ 5 ]. CRL4 consists of a Cullin4 (CUL4) scaffold that bridges the catalytic RING-domain subunit ROC1 to the adaptor protein DDB1, which in turn binds to exchangeable substrate receptors (DCAFs, DDB1- and CUL4-associated factors) [ 13 – 17 ]. In some instances, the DDB1 adaptor serves as an anchor for virus proteins, which then act as “viral DCAFs” to recruit the antiviral substrate. Examples are the simian virus 5 V protein and mouse cytomegalovirus M27, which bind to DDB1 and recruit STAT1/2 proteins for ubiquitylation, in order to interfere with the host’s interferon response [ 18 – 20 ]. Similarly, CUL4-dependent downregulation of STAT signalling is important for West Nile Virus replication [ 21 ]. In addition, the hepatitis B virus X protein hijacks DDB1 to induce proteasomal destruction of the structural maintenance of chromosome (SMC) complex to promote virus replication [ 22 , 23 ].

A large proportion of viruses have evolved means to co-opt their host’s ubiquitylation machinery, in order to improve replication conditions, either by introducing viral ubiquitin ligases and deubiquitinases, or by modification of host proteins involved in ubiquitylation [ 1 – 3 ]. In particular, host ubiquitin ligases are a prominent target for viral usurpation, to redirect specificity towards antiviral host restriction factors. This results in recruitment of restriction factors as non-endogenous neo-substrates, inducing their poly-ubiquitylation and subsequent proteasomal degradation [ 4 – 8 ]. This counteraction of the host’s antiviral repertoire is essential for virus infectivity and spread [ 9 – 12 ], and mechanistic insights into these specificity changes extend our understanding of viral pathogenesis and might pave the way for novel treatments.

Comparison of neo-substrate recognition modes of Vpr mus ( A ), Vpx sm ( B ), Vpx mnd2 ( C ) and Vpr HIV-1 ( D ). DCAF1-CtD is shown as grey cartoon and semi-transparent surface, Vpr mus −green, Vpx sm −orange, Vpx mnd2 –blue and Vpr HIV-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 Vpr mus −yellow, SAMHD1-CtD (bound to Vpx sm , PDB 4cc9) [ 50 ]–mint green, SAMHD1-NtD (Vpx mnd2 , PDB 5aja) [ 51 ]–magenta, UNG2 (Vpr HIV-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 Vpr mnd-2 amino acid sequence. Residues shaded grey or black are at least 60% or 90% type-conserved in all Vpx and Vpr proteins, respectively.

These data allow for structural comparison with neo-substrate binding modes of Vpx and Vpr proteins from different retrovirus lineages ( Fig 6A and 6B and 6C and 6D ). Vpx HIV-2 and Vpx sm position SAMHD1-CtD at the side of the DCAF1 BP domain through interactions with the N-termini of Vpx Helices-1 and -3 ( Fig 6B ) [ 50 ]. Vpx mnd2 and Vpx rcm bind SAMHD1-NtD using a bipartite interface comprising the side of the DCAF1 BP and the upper surface of the Vpx helix bundle ( Fig 6C ) [ 51 , 52 ]. Vpr HIV-1 engages its ubiquitylation substrate UNG2 using both the top and the upper edge of the Vpr HIV-1 helix bundle ( Fig 6D ) [ 54 ]. Of note, these upper-surface interaction interfaces only partially overlap with the Vpr mus /SAMHD1-CtD binding interface identified here and employ fundamentally different sets of interacting amino acid residues (Figs 6E and S6A ). Thus, it appears that the molecular interaction interfaces driving Vpx/Vpr-mediated neo-substrate recognition and degradation are not conserved between related SIV and HIV Vpx/Vpr accessory proteins, even in cases where identical SAMHD1-CtD regions are targeted for recruitment.

Taken together, the structural, biochemical and CLMS data are consistent with a model where the very C-terminus of SAMHD1 is recruited by Vpr mus , to place the remaining SAMHD1 domains appropriately for access to the catalytic machinery at the distal end of the CRL4 stalk.

In order to evaluate the distance information inherent in SAMHD1-CtD cross-links in a more quantitative way, the volume accessible to SAMHD1-CtD for interaction with CRL4 DCAF1-CtD /Vpr mus , consistent with the CLMS distance restraints, was simulated using the DisVis software tool [ 60 , 61 ]. For this analysis, SAMHD1-CtD was modelled as peptide in extended conformation. During the simulation, the state-2 CRL4 DCAF1-CtD /Vpr mus molecular model was kept fixed, and a six-dimensional search of all possible degrees of freedom of rotation and translation for the SAMHD1-CtD model in molecular contact with CRL4 DCAF1-CtD /Vpr mus was computed and ranked according to agreement with CLMS distance restraints. To visualise the output, all possible spatial positions of the centre of mass of SAMHD1-CtD, which satisfy >50% of the CLMS restraints, were plotted as density map on the structure of DCAF1-CtD/Vpr mus ( Fig 5C ). In accordance with the cryo-EM reconstruction, this independent computational analysis also locates SAMHD1-CtD on top of the Vpr mus helix bundle.

( A ) Schematic representation of sulfo-SDA cross-links between CRL4 DCAF1 /Vpr mus 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-NEDD8 DCAF1-CtD /Vpr mus /SAMHD1 (state-2), obtained from cryo-EM analysis ( Fig 3 ). SAMHD1-CtD density from the CRL4-NEDD8 DCAF1-CtD /Vpr mus /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 Vpr mus are oriented and coloured as in Fig 4A .

An additional 300 cross-links involved SAMHD1, extending to the C-terminal half of CUL4, to a DDB1 sequence stretch comprising amino acid residues 900–1000, to parts of DCAF1-CtD and to Vpr mus (Figs 5A and S5E ). The CRL4 DCAF1-CtD /Vpr mus residues exhibiting cross-links to SAMHD1 were mapped onto the state-2 model, and showed the presence of a large, yet defined, interaction surface ( Fig 5B ). Importantly, cross-links were apparent between the C-terminus of SAMHD1-CtD (residues K622, K626) and a region in Vpr mus Helix-1 (residues 27–36), which forms a part of the putative SAMHD1-CtD binding interface observed in cryo-EM, and which contains Vpr mus W29, one of the residues substituted in the mutagenesis and biochemical analysis presented above ( Fig 5B , purple spheres). In addition, amino acid residues from the N-terminal portion of SAMHD1-CtD (residues K595, K596, T602-S606) cross-linked close to the DCAF1-CtD “acidic loop” (residues 1092–1096), which is immobilised by Vpr near the proposed SAMHD1-CtD binding site, and to the very C-terminus of CUL4 (residues Y744, A759), which is also adjacent to the predicted SAMHD1-CtD binding position ( Fig 5B , pink spheres). Lastly, cross-links in the SAMHD1 N-terminal SAM and catalytic HD domains almost exclusively involved patches of the CRL4 DCAF1-CtD /Vpr mus surface surrounding and facing towards the putative SAMHD1-CtD attachment point ( Fig 5B , light brown spheres). These observations are compatible with recruitment of SAMHD-CtD on the upper surface of the Vpr helix bundle, as indicated by cryo-EM. In addition, the spatial distribution of cross-links involving the SAMHD1 N-terminal domains suggest that these are flexibly connected to SAMHD1-CtD, leading to highly variable positioning relative to CRL4 DCAF1-CtD /Vpr mus and thus offering a multitude of cross-linking opportunities to nearby CRL4 components, again in line with the cryo-EM reconstruction results, especially upon consideration of the positional heterogeneity of the CUL4 stalk ( Fig 3 ).

To obtain additional experimental evidence, the CRL4 DCAF1-CtD /Vpr mus /SAMHD1 assembly was further examined by cross-linking mass spectrometry (CLMS), using the photo-reactive cross-linker sulfo-SDA. This bi-functional compound contains an NHS-ester functional group on one end that reacts with primary amines and hydroxyl groups, while the other end covalently links to any amino acid sidechain within reach upon UV-activation via a carbene intermediate [ 58 ]. Accordingly, incubation of proteins or protein complexes with sulfo-SDA, followed by UV-illumination, allows for high-density cross-linking of lysine, and to a lesser extent serine, threonine and tyrosine side chains to amino acids within reach of the SDA spacer group, with faster kinetics than pure NHS ester-based cross-linkers, due to the short half-life of the UV-activated intermediate. Cross-linked peptides are subsequently identified by mass spectrometry, and provide insights into the topology and residue-residue distances of proteins and complexes [ 59 ]. In the case of the CRL4 DCAF1-CtD /Vpr mus /SAMHD1 assembly, the majority of identified cross-links that can be mapped onto the structure (468/519, 90.2%) are within the 25 Å violation threshold imposed by the geometry of the SDA spacer. Interestingly, 8 of the 11 cross-links between DDB1 and CUL4 are satisfied by the state-1 model, but increasingly violated in states-2 and -3 ( S5E Fig ), supporting in solution the rotational flexibility of the CRL4 stalk with respect to DDB1/DCAF1-CtD, as observed in cryo-EM ( Fig 3 ).

To test this hypothesis, Vpr mus amino acid residues in close proximity to the putative SAMHD1-CtD density were substituted by site-directed mutagenesis. Specifically, Vpr mus W29 was changed to alanine to block a hydrophobic contact with SAMHD1-CtD involving the aromatic side chain, and Vpr mus A66 was changed to a bulky tryptophan, in order to introduce a steric clash with SAMHD1-CtD ( Fig 4B ). The structural integrity of the Vpr mus W29A A66W double mutant was confirmed by CD spectroscopy ( S3H Fig ), and it was then assessed for complex formation with DDB1/DCAF1-CtD and SAMHD1 by analytical GF. In comparison to wild type Vpr mus , the W29A A66W mutant showed reduced DDB1/DCAF1-CtD/Vpr mus /SAMHD1 complex peak intensity ( Fig 4C , fraction 6), concomitant with (i) enrichment of DDB1/DCAF1-CtD/Vpr mus ternary complex, sub-stoichiometrically bound to SAMHD1 ( Fig 4C , fraction 7), (ii) excess DDB1/DCAF1-CtD complex ( Fig 4C , fraction 8), and (iii) excess SAMHD1 ( Fig 4C , fractions 9–10). In addition, binary combinations of the Vpr mus W29A A66W double mutant with DDB1/DCAF1-CtD or SAMHD1 were also analysed by GF. These data show loss of SAMHD1 interaction ( Fig 4D ), while the ability to bind DDB1/DCAF1-CtD is retained ( Fig 4E ). Together, these biochemical analyses support a location of the SAMHD1-CtD binding site on the upper surface of the Vpr mus helix bundle, as suggested by medium-resolution cryo-EM reconstruction.

( A ) Two views of the cryo-EM reconstruction of the CRL4-NEDD8 DCAF1-CtD /Vpr mus /SAMHD1 core. The crystal structure of the DDB1/DCAF1-CtD/Vpr mus 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 Vpr mus helix bundle. ( B ) Detailed view of the SAMHD1-CtD electron density. The model is in the same orientation as in A , left panel. Selected Vpr mus 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, Vpr mus or the Vpr mus 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 Vpr mus W29A/A66W ( D ) or DDB1/DCAF1-CtD and Vpr mus 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.

A reanalysis of the cryo-EM data involving template-based particle picking and extensive 3D classification allowed for separation of an additional homogeneous particle population ( S5A and S5B Fig ). This subset of particle images yielded a 3D reconstruction at a nominal resolution of 7.3 Å that only contained electron density corresponding to the CRL4 core (S5A and S5B and S5C and S5D Fig ). Molecular models of DDB1 BP domains A and C (BPA, BPC), DCAF1-CtD and Vpr mus , derived from our crystal structure ( Fig 2 ), could be fitted as rigid bodies into this cryo-EM volume ( Fig 4A ). No obvious electron density was visible for the bulk of SAMHD1. However, close inspection revealed an additional tubular, slightly arcing density feature, approx. 35 Å in length, located on the upper surface of the Vpr mus helix bundle, approximately 17 Å away from and opposite of the Vpr mus /DCAF1-CtD binding interface ( Fig 4A , red arrows). One end of the tubular volume contacts the middle of Vpr mus Helix-1, and the other end forms additional contacts to the C-terminus of Helix-2 and the N-terminus of Helix-3 ( Fig 4B ). A local resolution of 7.5–8 Å ( S5C Fig ) precluded the fitting of an atomic model. Considering the biochemical data, showing that SAMHD1-CtD is sufficient for recruitment to DDB1/DCAF1/Vpr mus , we hypothesise that this observed electron density feature corresponds to a region of SAMHD1-CtD which physically interacts with Vpr mus . Given its dimensions, the putative SAMHD1-CtD density could accommodate approx. 10 amino acid residues in a fully extended conformation or up to 23 residues in a kinked helical arrangement. All previous crystal structure analyses [ 46 ], as well as secondary structure predictions indicate that SAMHD1 residues C-terminal to the catalytic HD domain and C-terminal lobe (amino acids 599–626) are disordered in the absence of additional binding partners. Accordingly, the N-terminal globular domains of the SAMHD1 molecule might be flexibly linked to the C-terminal tether identified here. In that case, the bulk of SAMHD1 samples a multitude of positions relative to the DDB1/DCAF1-CtD/Vpr mus core, and consequently is averaged out in the process of cryo-EM reconstruction.

These data are in line with previous prediction based on extensive comparative crystal structure analyses, which postulated an approx. 150° rotation of the CRL4 stalk around the core [ 13 , 15 , 16 , 19 , 57 ]. However, the left- and rightmost CUL4 orientations observed here, states-1 and -3 from our cryo-EM analysis, indicate a slightly narrower stalk rotation range (119°), when compared to the outermost stalk conformations modelled from previously determined crystal structures (143°) ( Fig 3C ). A possible explanation for this discrepancy arises from inspection of the cryo-EM densities and fitted models, revealing that along with the main interaction interface on DDB1 BPB there are additional molecular contacts between CUL4 and DDB1. Specifically, in state-1, there is a contact between the loop connecting helices D and E of CUL4 cullin repeat (CR)1 (residues 161–169) and a loop protruding from BP blade 3 of DDB1 BPC (residues 795–801, S4I Fig ). In state-3, the loop between CUL4 CR2 helices D and E (residues 275–282) abuts a region in the C-terminal helical domain of DDB1 (residues 1110–1127, S4J Fig ). These auxiliary interactions might be required to lock the outermost stalk positions observed here in order to confine the rotation range of CUL4.

Alignment of 3D volumes from states-1, -2 and -3 shows that core densities representing DDB1 BPA, BPC, DCAF1-CtD and Vpr mus superimpose well, indicating that these components do not undergo major conformational fluctuations and thus form a rigid platform for substrate binding and attachment of the CRL4 stalk ( Fig 3 ). However, rotation of DDB1 BPB around a hinge connecting it to BPC results in three different orientations of state-1, -2 and -3 stalk regions relative to the core. BPB rotation angles were measured as 69° between state-1 and -2, and 50° between state-2 and -3.

( A ) Two views of an overlay of CRL4-NEDD8 DCAF1-CtD /Vpr mus /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 Vpr mus have been superimposed. ( B ) Two views of a superposition of DDB1/DCAF1-CtD/Vpr mus 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/Vpr mus 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).

Two consecutive rounds of 3D classification yielded three particle populations, resulting in 3D reconstructions at 8–10 Å resolution, which contained both the Vpr mus -bound CRL4 core and the stalk (conformational states-1, -2 and -3, Figs 3A and S4D and S4E and S4F ). The quality of the 3D volumes was sufficient to fit crystallographic models of core ( Fig 2 ) and the stalk (PDB 2hye) [ 15 ] as rigid bodies (Figs 3B and S4G ). For the catalytic RING-domain subunit ROC1, only fragmented electron density was present near the position it occupies in the crystallographic model ( S4G Fig ). In all three states, electron density was selectively absent for the C-terminal CUL4 winged helix B (WHB) domain (residues 674–759), which contains the NEDD8 modification site (K705), and for the preceding α-helix, which connects the CUL4 N-terminal domain to the WHB domain ( S4G Fig ). In accordance with this observation, the positions of CRL5-attached NEDD8 and of the CRL4 ROC1 RING domain are sterically incompatible upon superposition of their respective crystal structures ( S4H Fig ) [ 56 ].

To obtain structural insight into Vpr mus in the context of a complete CRL4 assembly, and to understand the SAMHD1 recruitment mechanism, we initiated cryo-EM analyses of the CRL4 DCAF1-CtD /Vpr mus /SAMHD1 assembly. In these studies, the small ubiquitin-like protein NEDD8 was enzymatically attached to the CUL4 subunit, in order to obtain its active form ( S4A Fig ) [ 55 ]. A CRL4-NEDD8 DCAF1-CtD /Vpr mus /SAMHD1 complex was reconstituted in vitro and purified by GF chromatography ( S4B Fig ). 2D classification of the resulting particle images revealed considerable compositional and conformational heterogeneity, especially regarding the presence and position of the CUL4-NEDD8/ROC1 sub-complex (stalk) relative to DDB1/DCAF1/Vpr mus (core) ( S4C Fig ).

To validate the importance of Vpr mus residues R15 and R75 for DCAF1-CtD-binding, charge reversal mutations to glutamates were generated by site-directed mutagenesis. The circular dichroism (CD) spectrum of the Vpr mus R15E R75E double mutant GST-fusion protein was identical to the wild type, indicating similar secondary structure content and thus no major structural disturbances caused by the amino acid substitutions ( S3H Fig ). The effect of the Vpr mus R15E R75E double mutant on complex assembly was then analysed by GF chromatography. SDS-PAGE analysis of the resulting chromatographic profile shows an almost complete loss of the DDB1/DCAF1-CtD/Vpr mus /SAMHD1 complex peak ( Fig 2D , fraction 6), when compared to the wild type, concomitant with enrichment of (i) some proportion of Vpr mus R15E R75E-bound DDB1/DCAF1-CtD ( Fig 2D , fraction 7), (ii) free DDB1/DCAF1-CtD (fraction 7–8), and of (iii) Vpr mus R15E R75E/SAMHD1 binary complex ( Fig 2D , fraction 8–9). This suggests that charge reversal of Vpr mus side chains R15 and R75 weakens the strong association with DCAF1 observed in wild type Vpr mus , due to loss of electrostatic interaction with the “acidic loop”, in accordance with the crystal structure. Consequently, some proportion of Vpr-bound SAMHD1 dissociates. This notion is further supported by GF analysis of binary combinations of the Vpr mus R15E R75E double mutant with either SAMHD1 or DDB1/DCAF1-CtD. Incubation of Vpr mus R15E R75E with SAMHD1 followed by GF leads to co-elution of both proteins, concomitant with a shift of the elution peak towards higher apparent molecular weight, compared to SAMHD1 alone ( Fig 2E , fractions 8–9). By contrast, incubation of the Vpr mus double mutant with DDB1/DCAF1-CtD does not change the elution volume of the DDB/DCAF1-CtD species, and no band corresponding to Vpr can be detected in the SDS-PAGE analysis of the corresponding fractions ( Fig 2F , fractions 7–8). These data clearly demonstrate loss of interaction with DDB1/DCAF1-CtD upon charge reversal of Vpr mus residues 15 and 75, while the SAMHD1-binding activity is retained.

Notably, in previously determined structures of Vpx/DCAF1/SAMHD1 complexes the “acidic loop” is a central point of ternary contact, providing a binding platform for positively charged amino acid side chains in either the SAMHD1 N- or C-terminus [ 50 – 52 ]. For example, Vpx sm positions SAMHD1-CtD in such a way, that SAMHD1 K622 engages in electrostatic interaction with the DCAF1 “acidic loop” residue D1092 ( Fig 2C , left panel). However, in the Vpr mus crystal structure the bound Vpr mus now blocks access to the corresponding SAMHD1-CtD binding pocket, in particular by the positioning of an extended N-terminal loop that precedes Helix-1. Additionally, Vpr mus side chains R15, R75 and R76 neutralise the DCAF1 “acidic loop”, precluding the formation of further salt bridges to basic residues in SAMHD1-CtD ( Fig 2C , right panel).

Superposition of the apo-DDB1/DCAF1-CtD and Vpr mus -bound crystal structures reveals conformational changes in DCAF1 upon Vpr mus association. Binding of the N-terminal arm of Vpr mus induces only a minor rearrangement of a loop in BP blade 2 ( S3C Fig ). By contrast, significant structural changes occur on the upper surface of the BP domain: polar and hydrophobic interactions of DCAF1 residues P1329, F1330, F1355, N1371, L1378, M1380 and T1382 with Vpr mus side chains of T79, R83, R86 and E87 in Helix-3 result in the stabilisation of the sequence stretch that connect BP blades 6 and 7 (“C-terminal loop”, Figs 2B and S3F ). Moreover, side chain electrostatic interactions of Vpr mus residues R15, R75 and R76 with DCAF1 E1088, E1091 and E1093 lock the conformation of an “acidic loop” upstream of BP blade 1, which is also unstructured and flexible in the absence of Vpr mus (Figs 2B and 2C and S3D and S3E and S3F ).

Vpr mus binds to the side and on top of the disk-shaped 7-bladed β-propeller (BP) DCAF1-CtD domain with a total contact surface area of ~1600 Å 2 comprising three major regions of interaction. The extended Vpr mus N-terminus attaches to the cleft between DCAF1 BP blades 1 and 2 through several hydrogen bonds, electrostatic and hydrophobic interactions (S3B and S3C and S3D Fig ). A second, smaller contact area is formed by hydrophobic interaction between Vpr mus residues L31 and E34 from Helix-1, and DCAF1 W1156, located in a loop on top of BP blade 2 ( S3E Fig ). The third interaction surface comprises the C-terminal half of Vpr mus Helix-3, which inserts into a ridge on top of DCAF1 ( S3F and S3G Fig ).

( A ) Overall structure of the DDB1/DCAF1-CtD/Vpr mus complex in two views. DCAF1-CtD is shown as grey cartoon and semi-transparent surface. Vpr mus 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 Vpr mus -bound DCAF1-CtD (grey/green cartoon). Only DCAF1-CtD regions with significant structural differences between apo- and Vpr mus -bound forms are shown. Disordered loops are indicated as dashed lines. ( C ) Comparison of the binary Vpr mus /DCAF1-CtD and ternary Vpx sm /DCAF1-CtD/SAMHD1-CtD complexes. For DCAF1-CtD, only the N-terminal “acidic loop” region is shown. Vpr mus , DCAF1-CtD and bound zinc are coloured as in A ; Vpx sm 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/Vpr mus or the Vpr mus 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 Vpr mus R15E/R75E ( E ) or DDB1/DCAF1-CtD and Vpr mus 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.

To obtain structural information regarding Vpr mus and its mode of binding to the CRL4 substrate receptor DCAF1, the X-ray crystal structures of a DDB1/DCAF1-CtD complex, and DDB1/DCAF1-CtD/T4L-Vpr mus (residues 1–92) fusion protein ternary complex were determined. The structures were solved using molecular replacement and refined to resolutions of 3.1 Å and 2.5 Å respectively ( S1 Table ). Vpr mus adopts a three-helix bundle fold, stabilised by coordination of a zinc ion by His and Cys residues on Helix-1 and at the C-terminus ( Fig 2A ). Superposition of Vpr mus with previously determined Vpx sm [ 50 ], Vpx mnd2 [ 51 , 52 ], and Vpr HIV-1 [ 54 ] structures reveals a conserved three-helix bundle fold, and similar position of the helix bundles on DCAF1-CtD ( S3A Fig ). In addition, the majority of side chains involved in DCAF1-interaction are type-conserved in all Vpx and Vpr proteins ( S3B and S3C and S3D and S3E and S3F and S3G and S6A Figs), strongly suggesting a common molecular mechanism of host CRL4-DCAF1 hijacking by the Vpx/Vpr family of accessory proteins. However, there are also significant differences in helix length and register as well as conformational variation in the loop region N-terminal of Helix-1, at the start of Helix-1 and in the loop between Helices-2 and -3 ( S3A Fig ).

To correlate these data with enzymatic activity, in vitro ubiquitylation assays were conducted by incubating SAMHD1, SAMHD1-ΔCtD or T4L-SAMHD1-CtD with purified CRL4 DCAF1-CtD , E1 (UBA1), E2 (UBCH5C), ubiquitin and ATP. Input proteins are shown in S2A Fig , and control reactions in S2B and S2C Fig . In the absence of Vpr mus , no SAMHD1 ubiquitylation was observed (Figs 1D and S2D ), while addition of Vpr mus resulted in robust SAMHD1 ubiquitylation, as demonstrated by an upward shift of SAMHD1 in the SDS PAGE analysis, induced by covalent modification with increasingly more ubiquitin molecules, leading to almost complete loss of the band corresponding to unmodified SAMHD1 after 15 min incubation (Figs 1E and S2E ). In agreement with the analytical GF data, SAMHD1-ΔCtD was not ubiquitylated in the presence of Vpr mus (Figs 1F and S2F ). By contrast, T4L-SAMHD1-CtD was efficiently ubiquitylated, resulting in >90% loss of the band corresponding to unmodified T4L-SAMHD1-CtD after 15 min (Figs 1G and S2F ). Again, these data substantiate the functional importance of SAMHD1-CtD for Vpr mus -mediated recruitment to the CRL4 DCAF1 ubiquitin ligase.

Previous cell-based assays indicated that residues 583–626 of rhesus macaque SAMHD1 (SAMHD1-CtD) are necessary for Vpr mus -induced proteasomal degradation [ 49 ]. To test this finding in our in vitro system, constructs containing SAMHD1-CtD fused to T4 lysozyme (T4L-SAMHD1-CtD), or containing only the N-terminal domains of SAMHD1, and lacking SAMHD1-CtD (SAMHD1-ΔCtD), were incubated with Vpr mus and DDB1/DCAF1-CtD, and complex formation was assessed by GF chromatography. Analysis of the resulting chromatograms by SDS-PAGE shows that SAMHD1-ΔCtD did not co-elute with DDB1/DCAF1-CtD/Vpr mus ( Fig 1B ). By contrast, T4L-SAMHD1-CtD accumulated in the same elution peak as DDB1/DCAF1-CtD and Vpr mus ( Fig 1C ). These results confirm that SAMHD1-CtD is necessary for stable association with DDB1/DCAF1-CtD/Vpr mus in vitro, and demonstrate that SAMHD1-CtD is sufficient for Vpr mus -mediated recruitment of the T4L-SAMHD1-CtD fusion construct to DDB1/DCAF1-CtD.

( A-C ) GF analysis of in vitro reconstitution of protein complexes containing DDB1/DCAF1-CtD, Vpr mus 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 Vpr mus , with the indicated SAMHD1 constructs as substrate. Reactions were stopped after the indicated times, separated on SDS-PAGE and visualised by Coomassie blue staining.

To investigate the molecular interactions between Vpr mus , the neo-substrate SAMHD1 from rhesus macaque and CRL4 subunits DDB1/DCAF1 C-terminal domain (DCAF1-CtD), protein complexes were reconstituted in vitro from purified components and analysed by gel filtration (GF) chromatography. The different protein constructs that were employed are shown schematically in S1A Fig . In the absence of additional binding partners, Vpr mus is insoluble after removal of the GST affinity purification tag ( S1B Fig ) and accordingly could not be applied to the GF column. No interaction of SAMHD1 with DDB1/DCAF1-CtD could be detected in the absence of Vpr mus ( S1C Fig ). Analysis of binary protein combinations (Vpr mus and DDB1/DCAF1-CtD; Vpr mus and SAMHD1) shows that Vpr mus elutes together with DDB1/DCAF1-CtD ( S1D Fig ) or with SAMHD1 ( S1E Fig ). Incubation of Vpr mus with DDB1/DCAF1B and SAMHD1 followed by GF resulted in co-elution of all three components ( Fig 1A ). Together, these results show that Vpr mus forms stable binary and ternary protein complexes with DDB1/DCAF1-CtD and/or SAMHD1 in vitro. Furthermore, incubation with any of these interaction partners apparently stabilises Vpr mus by alleviating its tendency for aggregation/insolubility.

Discussion

Our X-ray crystallographic studies of the DDB1/DCAF1-CtD/Vpr mus assembly provide the first structural insight into a class of “hybrid” SIV Vpr proteins. These are present in the SIV agm and SIV mus/deb/syk lineages of lentiviruses and combine characteristics of related Vpr HIV-1 and SIV Vpx accessory proteins.

Like SIV Vpx, “hybrid” Vpr proteins down-regulate the host restriction factor SAMHD1 by recruiting it to CRL4DCAF1 for ubiquitylation and subsequent proteasomal degradation. However, using a combination of X-ray, cryo-EM and CLMS analyses, we show that the molecular strategy, which Vpr mus evolved to target SAMHD1, is strikingly different from Vpx-containing SIV strains. In the two clades of Vpx proteins, divergent amino acid sequence stretches just upstream of Helix-1 (variable region (VR)1, S6A Fig), together with polymorphisms in the SAMHD1-N-terminus of the respective host species, determine if HIV-2-type or SIV mnd -type Vpx recognise SAMHD1-CtD or SAMHD1-NtD, respectively. These recognition mechanisms result in positioning of SAMHD1-CtD or -NtD on the side of the DCAF1 BP domain in a way that allows for additional contacts between SAMHD1 and DCAF1, thus forming ternary Vpx/SAMHD1/DCAF1 assemblies with very low dissociation rates [50–52,62]. In Vpr mus , different principles determine the specificity for SAMHD1-CtD. Here, VR1 is not involved in SAMHD1-CtD-binding at all, but forms additional interactions with DCAF1, which are not observed in Vpx/DCAF1 protein complexes (S6A Fig). Molecular contacts between Vpr mus and SAMHD1 are dispersed on Helices-1 and -3, facing away from the DCAF1 interaction site and immobilising SAMHD1-CtD on the top side of the Vpr mus helix bundle (S6A Fig). Placement of SAMHD1-CtD in such a position precludes stabilising ternary interaction with DCAF1-CtD, but still results in robust SAMHD1 ubiquitylation in vitro and SAMHD1 degradation in cell-based assays [24]. Accordingly, our in vitro reconstitution analyses show that Vpr mus is able to form stable binary interaction with either SAMHD1 or DCAF1-CtD, in the absence of the respective third binding partner. This leaves the question unanswered, if in a physiological setting, upon host cell infection, Vpr mus first captures SAMHD1 to guide it to the CRL4 complex, or if it hijacks CRL4 to subsequently recruit SAMHD1. However, since CRL4 localises to both cytoplasm and nucleus [63], while SAMHD1 is exclusively found in the nucleus [64], it is tempting to speculate that upon entering the host cell, Vpr at first encounters and binds cytoplasmic CRL4DCAF1, to subsequently translocate into the nucleus for SAMHD1 recruitment.

Predictions regarding the molecular mechanism of SAMHD1-binding by other “hybrid” Vpr orthologues are difficult due to sequence divergence. Even in Vpr deb , the closest relative to Vpr mus , only approximately 50% of amino acid side chains lining the putative SAMHD1-CtD binding pocket are conserved (S6A Fig). Previous in vitro ubiquitylation and cell-based degradation experiments did not show a clear preference of Vpr deb for recruitment of either SAMHD1-NtD or–CtD [24,49]. Furthermore, it is disputed if Vpr deb actually binds DCAF1 [65], which might possibly be explained by amino acid variations in the very N-terminus and/or in Helix-3 (S6A Fig). Vpr syk is specific for SAMHD1-CtD [49], but the majority of residues forming the binding platform for SAMHD1-CtD observed in the present study are not conserved. The SIV agm lineage of Vpr proteins is even more divergent, with significant differences not only in possible SAMHD1-contacting residues, but also in the sequence stretches preceding Helix-1, and connecting Helices-2 and -3, as well as in the N-terminal half of Helix-3 (S6A Fig). Furthermore, there are indications that recruitment of SAMHD1 by the Vpr agm.GRI sub-type involves molecular recognition of both SAMHD1-NtD and–CtD [49,53]. In conclusion, recurring rounds of evolutionary lentiviral adaptation to the host SAMHD1 restriction factor, followed by host re-adaptation, resulted in highly species-specific, diverse molecular modes of Vpr-SAMHD1 interaction. Similar molecular arms races between cell-intrinsic antiviral host factors and viral antagonists shaped the species-specific lentiviral antagonism of e.g. host restriction factors of the APOBEC3 family and tetherin, through induction of their degradation by the respective viral antagonists Vif or Nef/Vpu [66–68]. Furthermore, viral re-adaptation to certain simian and human variants of these restriction factors, following cross-species transmission, took part in the emergence of pandemic HIV strains, thus highlighting the importance of structural insight into these processes [9]. In addition to the instance presented here, further structural characterisation of SAMHD1-Vpr complexes will be necessary to fully define outcomes of this particular virus-host molecular arms race.

Previous structural investigation of DDB1/DCAF1/Vpr HIV-1 in complex with the neo-substrate UNG2 demonstrated that Vpr HIV-1 engages UNG2 by mimicking the DNA phosphate backbone. More precisely, UNG2 residues, which project into the major groove of its endogenous DNA substrate, insert into a hydrophobic cleft formed by Vpr HIV-1 Helices-1, -2 and the N-terminal half of Helix-3 [54]. This mechanism might rationalise Vpr HIV-1 ’s extraordinary binding promiscuity, since the list of potential Vpr HIV-1 degradation substrates is significantly enriched in DNA- and RNA-binding proteins [27]. Moreover, promiscuous Vpr HIV-1 -induced degradation of host factors with DNA- or RNA-binding activity has been proposed to induce cell cycle arrest at the G2/M phase border, which is the most thoroughly described phenotype of Vpr proteins so far [26,27,69]. In Vpr mus , the N-terminal half of Helix-1 as well as the bulky amino acid residue W48, which is also conserved in Vpr agm and Vpx, constrict the hydrophobic cleft (S6A and S6B Fig). Furthermore, the extended N-terminus of Vpr mus Helix-3 is not compatible with UNG2-binding due to steric exclusion (S6C Fig). In accordance with these observations, Vpr mus does not down-regulate UNG2 in a human T cell line [27]. However, Vpr mus , Vpr syk and Vpr agm also cause G2/M cell cycle arrest in their respective host cells [65,70,71]. This strongly hints at the existence of further structural determinants in Vpr mus , Vpr syk , Vpr agm and potentially Vpr HIV-1 , which regulate recruitment and ubiquitylation of DNA/RNA-binding host factors, in addition to the hydrophobic, DNA-mimicking cleft on top of the three-helix bundle. Future efforts to structurally characterise these determinants will further extend our understanding of how the Vpx/Vpr helical scaffold binds, and in this way adapts to a multitude of neo-substrate epitopes.

Our cryo-EM reconstructions of CRL4DCAF1-CtD/Vpr mus /SAMHD1, complemented by CLMS, also provide insights into the structural dynamics of CRL4 assemblies prior to ubiquitin transfer. The data confirm previously described rotational movement of the CRL4 stalk, in the absence of constraints imposed by a crystal lattice, creating a ubiquitylation zone around the Vpr mus -modified substrate receptor (Figs 3 and 7A) [13,15,16,19,57]. Missing density for the neddylated CUL4 WHB domain and for the catalytic ROC1 RING domain indicates that these distal stalk elements are highly mobile and likely sample a multitude of orientations relative to the CUL4 scaffold (S4G Fig). These observations are in line with structure analyses of CRL1 and CRL5, where CUL1/5 neddylation leads to re-orientation of the cullin WHB domain, and to release of the ROC1 RING domain from the cullin scaffold, concomitant with stimulation of ubiquitylation activity [56]. Moreover, recent cryo-EM structure analysis of CRL1β-TRCP/IκBα demonstrated substantial mobility of pre-catalytic CUL1-NEDD8 WHB and ROC1 RING domains [72]. Such flexibility seems necessary to structurally organise multiple CRL1-dependent processes, in particular the nucleation of a catalytic assembly, involving intricate protein-protein interactions between NEDD8, CUL1, ubiquitin-charged E2 and substrate receptor. This synergistic assembly then steers the ubiquitin C-terminus towards a substrate lysine for priming with ubiquitin [72]. Accordingly, our cryo-EM studies might indicate that similar principles apply for CRL4-catalysed ubiquitylation. However, to unravel the catalytic architecture of CRL4, sophisticated cross-linking procedures as in reference [72] will have to be pursued.

The mobility of the CRL4 stalk might assist the accommodation of a variety of sizes and shapes of substrates in the CRL4 ubiquitylation zone and might rationalise the wide substrate range accessible to CRL4 ubiquitylation through multiple DCAF receptors (Fig 7A). Owing to selective pressure to counteract the host’s SAMHD1 restriction, HIV-2 and certain SIVs have taken advantage of this dynamic CRL4 architecture by modification of the DCAF1 substrate receptor with Vpx/Vpr-family accessory proteins. In such a way, either SAMHD1-CtD or -NtD is tethered to DCAF1, in order to flexibly recruit the bulk of SAMHD1 to further improve the accessibility of lysine side chains both tether-proximal and on the SAMHD1 globular domains to the neddylated CRL4 catalytic assembly (Fig 7B and 7C).

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larger image TIFF original image Download: Fig 7. Schematic illustration of structural plasticity in Vpr mus -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 Vpr mus 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. https://doi.org/10.1371/journal.ppat.1009775.g007

The catalytic dNTP triphosphohydrolase activity of SAMHD1 depends on nucleotide-dependent oligomerisation, mediated by two allosteric nucleotide-binding sites. In the absence of nucleotides, SAMHD1 adopts a monomer-dimer equilibrium with an equilibrium dissociation constant in the low micromolar range [73]. In the present work, SAMHD1 preparations and subsequent biochemical and structural studies have been performed without exogenously added nucleotides. Hence, under the experimental conditions, monomeric and dimeric states of SAMHD1 are expected to co-exist, competent for recruitment to Vpr. For clarity, only the binding of a monomeric SAMHD1 species is schematically indicated in Fig 7. However, recruitment of a SAMHD1 dimer might expose additional surface-exposed lysine residues to the CRL4 catalytic machinery and thus might further improve the efficacy of SAMHD1 ubiquitylation.

Insertion of guanine-based nucleotides in the first binding site shifts the SAMHD1 monomer-dimer equilibrium towards the dimeric form, and dNTP-binding to the second site leads to assembly of the catalytically active tetramer [41,73–78]. In accordance with the absence of such nucleotides, our analytical gel filtration data and cryo-EM reconstructions do not support the existence of SAMHD1 tetramers under the experimental conditions (Figs 1 and S1 and S4 and S5). However, SAMHD1-CtD is essential for tetramer formation by contributing critical molecular contacts to neighbouring protomers [46]. Furthermore, tetramer destabilisation by CDK1/2-cyclinA-dependent phosphorylation of T592 in SAMHD1-CtD endogenously attenuates SAMHD1 activity in cycling cells [46,77,79,80]. Hence, under physiological conditions, it is conceivable that Vpr mus destabilises SAMHD1 tetramers by sequestering SAMHD1-CtD, in order to abrogate SAMHD1 activity, prior to inducing its proteasomal degradation. Such a mechanism would be in accordance with previous observation of Vpx HIV-2 -mediated SAMHD1 tetramer disassembly and inhibition of dNTPase activity [62].

Altogether, intrinsic CRL4 mobility, in combination with flexible Vpx/Vpr-mediated SAMHD1 recruitment maximises the efficiency of SAMHD1 poly-ubiquitylation and proteasomal degradation to stimulate virus replication (Fig 7D). In infected cells however, there is a stoichiometric mismatch between less than 1000 Vpr molecules, which are introduced in the host cell, and SAMHD1, which is abundant across a broad range of tissues and cell types [26,81]. Tight coupling of CRL4 to the p97 ATPase confers efficient unfolding of poly-ubiquitylated substrates prior to proteasomal degradation [82]. In this way, ubiquitylated SAMHD1 is removed from Vpr-bound CRL4DCAF1 to initiate subsequent rounds of SAMHD1 recruitment, ubiquitylation and degradation.

[END]

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