Structural Basis of Clade-specific Engagement of SAMHD1 (Sterile α Motif and Histidine/Aspartate-containing Protein 1) Restriction Factors by Lentiviral Viral Protein X (Vpx) Virulence Factors*

Background: Lentiviral Vpx binding to primate SAMHD1 is under positive selection. Results: Different Vpx protein variants interact with the N-terminal domain or the C-terminal tail of SAMHD1 in ubiquitin-ligase-substrate receptor complexes in a unique fashion. Conclusion: Vpx antagonizes SAMHD1 by recruiting it via two separate regions for proteasomal degradation. Significance: Our findings shed light on how lentivirus virulence factors intersect with host innate immunity. Sterile α motif (SAM) and histidine/aspartate (HD)-containing protein 1 (SAMHD1) restricts human/simian immunodeficiency virus infection in certain cell types and is counteracted by the virulence factor Vpx. Current evidence indicates that Vpx recruits SAMHD1 to the Cullin4-Ring Finger E3 ubiquitin ligase (CRL4) by facilitating an interaction between SAMHD1 and the substrate receptor DDB1- and Cullin4-associated factor 1 (DCAF1), thereby targeting SAMHD1 for proteasome-dependent down-regulation. Host-pathogen co-evolution and positive selection at the interfaces of host-pathogen complexes are associated with sequence divergence and varying functional consequences. Two alternative interaction interfaces are used by SAMHD1 and Vpx: the SAMHD1 N-terminal tail and the adjacent SAM domain or the C-terminal tail proceeding the HD domain are targeted by different Vpx variants in a unique fashion. In contrast, the C-terminal WD40 domain of DCAF1 interfaces similarly with the two above complexes. Comprehensive biochemical and structural biology approaches permitted us to delineate details of clade-specific recognition of SAMHD1 by lentiviral Vpx proteins. We show that not only the SAM domain but also the N-terminal tail engages in the DCAF1-Vpx interaction. Furthermore, we show that changing the single Ser-52 in human SAMHD1 to Phe, the residue found in SAMHD1 of Red-capped monkey and Mandrill, allows it to be recognized by Vpx proteins of simian viruses infecting those primate species, which normally does not target wild type human SAMHD1 for degradation.

Host-pathogen co-evolution leads to positive selection, the degree of which can be identified through interspecies sequence alignments (21,22). Positive selection at the interfaces of host-pathogen complexes is present if the ratio of nonsynonymous changes to the number of synonymous changes per site is greater than 1 (23). Thus, interfaces that are used in the interaction between host and pathogen proteins accumulate more frequent amino acid changes than would occur at random. Evolutionary signatures resulting from positive selection permit differentiation of broad acting anti-viral proteins from narrowly effective virus-specific antagonists. In particular, strong positive selection signals at several sites in a single host gene suggest that the gene product is targeted by different viruses at multiple regions. This is noted for SAMHD1, in which two separate regions, the SAM domain and the C terminus, interact with Vpx from distinct HIV/SIV strains. For example, Vpx proteins from the clade that includes HIV-2 and SIVsm (isolated from the sooty mangabey monkey) target a sequence element in the C terminus of SAMHD1, whereas those from the clade that includes SIVrcm (isolated from the Red-capped monkey (RCM)) and SIVmnd (isolated from the Mandrill monkey) interact with the SAM domain, with key residues mapping to different sites of the protein (20, 24 -29).
Here, we investigated the positioning and distribution of selection signals using biochemical and structural approaches to elucidate the molecular basis of Vpx/SAMHD1 co-evolution. Our data show that the large N-terminal region of MND SAMHD1, including the SAM domain, interacts with Vpx SIVrcm and Vpx SIVmnd in the context of DCAF1. Changing several amino acids residues in the N-terminal region of human (Hu) SAMHD1 to those found at equivalent positions in the simian proteins revealed that substitution of only a single residue, Ser-52 to Phe, is sufficient to allow recognition by SIVrcm and SIVmndVpx of and recruitment to the CRL4-DCAF1 E3 ubiquitin ligase, whereas most other changes were invariant for recruitment. Our combined structural and biochemical results provide important molecular insights into how Vpx proteins from different HIV/SIV clades engage different elements in the cellular SAMHD1 protein to overcome the cell's antiviral defense.

Experimental Procedures
Cloning and Plasmid Construction-The N-terminal regions (NTD, residues 1-115) of Hu, RCM, and MND SAMHD1 were cloned into pET41 (EMD Biosciences) with GST and His 6 tags at their N termini and C termini, respectively. The NTDs were also cloned into pET21 (EMD Biosciences) with His 6 tags at their C termini. The C terminus of RCM SAMHD1 (residues 595-626, CTD) was cloned into a modified pET32 vector containing a tobacco etch virus protease site at the C terminus of His 6 -tagged thioredoxin fusion. Full-length Vpx SIVrcm (residues 1-108) or residues 1-90 (Vpx(⌬C) SIVrcm ) from redcapped monkey were cloned into pET43 vectors with a C-terminal His 6 tag modified to include a tobacco etch virus protease site between the N-terminal NusA fusion the Vpx proteins, as described previously (20). Full-length Vpx SIVmnd (residues 1-99) and residues 1-89 (Vpx(⌬C) SIVmnd ) were also cloned into pET43 vector as described above. DCAF1, human and monkey SAMHD1, Vpx SIVmnd , and Vpx SIVrcm were also cloned, separately, into pCDNA with a HA tag at the N terminus. The cDNAs for monkey SAMHD1, Vpx SIVmnd , and Vpx SIVrcm were provided by M. Emerman (Fred Hutchison Cancer Center, Seattle, WA) (25,27). Site-specific mutants of Hu and RCM SAMHD1 were prepared using the QuikChange mutagenesis kit (Agilent Technologies). All other clones were described previously (30).
Isothermal Titration Calorimetry-Typically, protein complexes, DDB1-DCAF1 CA -Vpx SIVrcm , were placed in the sample cell of the calorimeter (MicroCal Inc., Northampton, MA), and aliquots of SAMHD1 NTD or CTD were added at 25°C. Titrations were carried out in 10 mM HEPES buffer, pH 8.0, 1 mM Tris(2-carboxyethyl)phosphine, 100 mM NaCl, and 0.02% sodium azide. All samples were dialyzed against this buffer and concentrated using Amicon concentrators (Millipore) before titrations. Titration of SAMHD1 NTD or CTD into the buffer was used for base-line correction. Data analyses were performed using Origin 7 software (OriginLab Corp).
X-ray Crystallography-Crystallization of the ternary complex consisting of DCAF1 CA , Vpx(⌬C) SIVmnd , and MND SAMHD1 NTD was carried out at room temperature by the sitting drop vapor diffusion method using drops comprising 2 l of complex (ϳ2 mg/ml) and 2 l of reservoir solution (1.6 M NaH 2 PO 4 , 0.4 M K 2 HPO 4 , 0.1 M sodium phosphate/citrate buffer, pH 4.2). A well diffracting crystal was obtained after ϳ2 weeks. X-ray diffraction data were collected on the flash-cooled (Ϫ180°C) crystal at the SER-CAT facility sector 22-BM beamline at the Advance Photon Source at Argonne National Laboratory, Chicago, IL, up to 2.6 Å resolution. All diffraction data were processed, integrated, and scaled using XDS (31). The crystallographic phase was determined by molecular replacement using the coordinates of DCAF1 and Vpx SIVsm in the ternary complex of DCAF1-Vpx-SAMHD1 CTD (PDB code 4CC9) (29), and the coordinates of the SWISS-MODEL-generated SAM domain model (32) based on the solution structure of the N terminus (residues 23-118) of SAMHD1 (PDB code 2E8O) (33) as structural probes in PHASER (34). After generation of the initial model, the chain was rebuilt using the program Coot (35). Iterative refinement was carried out by alternating between manual re-building in Coot (35), refinement in BUSTER (36), and refinement in PHENIX (37). The final model, refined in PHENIX, exhibited clear electron density for residues Arg-1073-Gln-1314 and Ser-1328 -Gly1390 of DCAF1, Glu-10 -Cys-88 of Vpx SIVmnd , and Gln-2-Arg-20 and Gly-35-Ser-107 (or Gln-2-Thr-21 and Gly-35-Gln-109) of MND SAMHD1 NTD, with an R-factor of 22.9% and a free R of 28.0%. Note that the electron density for backbone atoms of MND SAMHD1 NTD residues residing in helices ␣3 and ␣4 were traceable but that of side chains was weakly observed. Of all the residues, a total of 97% were located in both favored and allowed regions of the Ramachandran plot, respectively, as evaluated by MOLPROBITY (38). A summary of pertinent data collection and refinement is provided in Table 1.
GST Pulldown Assays-Typically, 100 pmol of GST-tagged SAMHD1 NTD was incubated with 100 pmol of DDB1-DCAF1 CA -Vpx in 250 l of buffer containing 25 mM sodium phosphate, pH 7.5, 150 mM NaCl, 2 mM DTT, 10% glycerol, 0.02% sodium azide, 0.3% Nonidet P-40, and 1% Tween (binding buffer) for 30 min with rocking at 4°C. Glutathione-Sepharose 4B beads (50 l of a 50% slurry) were added to the protein mixtures and further agitated for 1 h. The beads were washed 4 times with 1 ml of binding buffer, and bound proteins were eluted with 50 l of Laemmli sample buffer, separated by 4 -20% SDS-PAGE, transferred to PVDF membrane, and detected by immunoblotting with anti-His antibody (Millipore).
In Vitro Ubiquitination Assays-Ubiquitination of Hu SAMHD1 wild type (WT) and the S52F mutant proteins were carried out using CRL4-DCAF1 CA -Vpx(⌬C) SIVrcm as the E3 ubiquitin ligase in the presence of UBA1, UbcH5, and ubiquitin as reported previously (20). The degree of ubiquitination was assessed by immunoblotting and detection with anti-T7 antibody (EMD Biosciences).

Results
Vpx SIVrcm Facilitates the Interaction between SAMHD1 and DDB1-DCAF1 via the N-terminal Domain of SAMHD1-We and others previously reported that Vpx SIVsm and Vpx HIV-2 preferentially interact with the CTD of SAMHD1, whereas Vpx SIVrcm and Vpx SIVmnd interact with the NTD of SAMHD1 ( Fig. 1A) (20,(25)(26)(27)(28). The interactions between these particular Vpx and the respective SAMHD1 proteins require the substrate  (25), we speculated that the NTD of RCM and MND SAMHD1 share a sequence motif that is tar-geted by Vpx SIVrcm and Vpx SIVmnd . To identify this motif, we aligned the amino acid sequences of the NTD of Hu, RCM, and MND SAMHD1 proteins (Fig. 1A). Despite being highly homologous, several residues, including amino acids at positions 3, 7, 15, 24, 32, 52, 60, 63, 72, 85, 107, and 108 are different between the monkey and human sequences. The two monkey sequences, on the other hand, contain only one conservative change at position 36 (Leu to Val). Thus, the NTD of RCM SAMHD1 and MND SAMHD1 are essentially identical. We, therefore, hypothesized that DDB1-DCAF1 CA -Vpx SIVrcm could bind to the NTD of both RCM and MND SAMHD1. To quantitatively determine binding affinities between SAMHD1 and DDB1-DCAF1-Vpx SIVrcm , we performed isothermal titration calorimetry (Fig. 1B). As expected, both the NTDs of RCM and MND SAMHD1 bound tightly to DDB1-DCAF1-Vpx SIVrcm , with K d values of 170 Ϯ 22 nM and 22 Ϯ 2 nM, respectively. In contrast, the CTD of RCM SAMHD1, which is identical to that of MND SAMHD1, did not show any measurable interaction with DDB1-DCAF1-Vpx SIVrcm . These isothermal titration calorimetry binding data quantitatively confirm our previous results obtained by analytical gel filtration analysis (27). Furthermore, the data suggest that Vpx SIVmnd and Vpx SIVrcm contain a common sequence motif(s) for recruitment of the NTD of primate SAMHD1 proteins. Indeed, the amino acid sequences of Vpx SIVmnd and Vpx SIVrcm are highly homologous, and the sequence motif, essential for recruitment of the C terminus of SAMHD1 to DCAF1, is not present in either of the two Vpx proteins (Fig. 1C). Structural Mapping of DCAF1-Vpx SIVmnd and DCAF1-Vpx SIVrcm Binding to the SAM Domain of SAMDH1-To identify the region of the SAM domain involved in the binding by DCAF1-Vpx SIVmnd or DCAF1-Vpx SIVrcm , we utilized NMR spectroscopy. Fig. 2  Residues whose resonances experience chemical shift changes upon binding (e.g. Gln-3, Ser-24, Val-36, Phe-52, Gly-60, and Gly-108 resonances, marked with solid ovals in Fig. 2) are indicated on the SAM domain structure (amino acids 23-118) of Hu SAMHD1 (PDB code 2E8O), with changes colored in red (Ͼ0.15 ppm) and orange (between 0.07 and 0.15 ppm). As can be easily appreciated, binding involves one face of the structure, forming a large extended binding site for DCAF1-Vpx SIVmnd (Fig. 2). Several of the affected residues are those that are different between monkey and human SAM (Fig. 1A), some of which reside on ␣ helices ␣1 and ␣2. However, not all divergent residues are affected by the DCAF1-Vpx-SIVmnd binding, in particular those residing on ␣ helices ␣3 and ␣4. For example, His-85 in the monkey sequences is replaced by Arg-85 in human (Fig. 1A), but its amide resonance exhibited negligible (Ͻ0.035 ppm) chemical shift changes upon complex formation (dashed oval in Fig. 2), demonstrating that His-85 and the surrounding region are not part of the binding interface.
In our NMR analysis, we also obtained information on the 30-residue N-terminal tail of the SAM domain that is flexible in the solution structure. The associated amide resonances appear

Distinct Modes of SAMHD1 Recruitment to CRL4-DCAF1 by Vpx
as sharp and intense cross-peaks in the central, poorly dispersed, random coil region of the 1 H, 15 N TROSY-HSQC spectrum of the free protein (blue resonances, enclosed by the dashed rectangle in Fig. 2). Interestingly, many of these N-terminal tail resonances exhibited large chemical shift perturbations upon binding DCAF1-Vpx SIVmnd , indicating that the N-terminal region is involved in complex formation and changes conformation in the complex.
A similar NMR study, mapping the binding of DCAF1-Vpx SIVrcm to MND SAMHD1 NTD, shows that essentially the same regions of the SAM domain are involved in both complexes (data not shown). Note that the MND SAMHD1 and RCM SAMHD1 exhibit identical amino acid sequences for the SAM domain, except for the L36V change.
Crystal Structure of the DCAF1-Vpx SIVmnd -SAMHD1 NTD Complex-To further delineate the details of SAMHD1 recruitment via its NTD for degradation by Vpx SIVmnd , we determined the crystal structure of the DCAF1-Vpx SIVmnd -SAMHD1 NTD complex at 2.6 Å resolution. Two ternary complexes are found in the asymmetric unit (data not shown), and given their near identity, only one is described below. Several important features are noted: two critical anchoring points are used by the SAMHD1 NTD to interact with DCAF1 and Vpx, involving the N-terminal tail (residues Gln-2-Arg-20) and the core SAM domain, in particular residues residing in helices ␣1 and ␣2. The N-terminal tail up to residue Arg-20 contacts both DCAF1 and Vpx residues and is intertwined between these two proteins (Fig. 3, A and B), whereas residues Gly-35-His-39 and the first two helices (␣1, residues Gly-46 -Gly-57, and ␣2, residues Gly-60 -Glu-70) of the SAMHD1 NTD interact solely with Vpx (Fig. 3A). This mode of interaction is different from what was observed in the structure of the DCAF1-Vpx SIVsm -SAMHD1 CTD (Fig. 3C) (29). On the other hand, despite sequence differences between Vpx SIVmnd and Vpx SIVsm (Fig. 1C), both Vpx proteins interact with DCAF1 in a very similar way (Fig. 3D) for recruitment of SAMHD1 to DCAF1 via the SAMHD1 NTD and CTD, respectively.
The above structural observations were validated in co-immunoprecipitation experiments. In particular, Vpx SIVmnd coimmunoprecipitated with MND SAMHD1 and WT DCAF1 as well as the F1330A/F1355A mutant DCAF1, whereas Vpx HIV-2 failed to immunoprecipitate Hu SAMHD1 with the F1330A/ F1355A DCAF1 mutant (Fig. 3E). On the other hand, the D1092A/E1093A DCAF1 mutation abolished both Vpx SIVmnd -SAMHD1 and Vpx HIV-2 -SAMHD1 interactions (Fig. 3E). These co-immunoprecipitation results can be explained based on the DCAF1-Vpx SIVmnd interface in the crystal structure. In the structure, the side chain of DCAF1 Asp-1092 is hydrogenbonded to the backbone amide of Phe-15 and the N⑀ of the Arg-14 side chain of MND SAMHD1 NTD as well as to the hydroxyl group of the Tyr-62 side chain of Vpx SIVmnd , whereas the side chain of DCAF1 Glu-1093 hydrogen bonds with the NH 2 group of Arg-66 and the N group of Lys-73 of MND SAMHD1 NTD (Fig. 3F). Similar hydrogen-bonding patterns are present in the DCAF1-Vpx SIVsm structure (29). Therefore, changing Asp-1092 and Glu-1093 to Ala eliminates these H-bonds (Fig. 3F). Similarly, residues Phe-1330 and Phe-1335 of DCAF1 engage in hydrophobic contacts with Tyr-76 in Vpx SIVmnd (Fig. 3F). The same hydrophobic triad can be formed with Phe-80 in Vpx HIV-2 (29), and removal of the two aromatic side chains on DCAF1 will abrogate the interaction with Vpx HIV-2 . This loss may possibly be compensated for in Vpx SIVmnd , where the hydroxyl group of the equivalent tyrosine is close to the O␥ hydroxyl group of Thr-1097 in DCAF1 (Fig.  3G). All together, the structural data clearly visualize how the distinct regions of SAMHD1 are targeted by clade-specific Vpx proteins.
A Single Residue Change in the Human SAMHD1 NTD Mediates Proteasome-dependent Down-regulation by Vpx SIVrcm -To identify the human SAMHD1 residue(s) that is responsible for the lack of recognition by Vpx SIVrcm and Vpx SIVmnd , N-terminal SAMHD1 residues that varied between human and monkey were exchanged, and GST pulldown assays with mixtures of GST-Hu SAMHD1 NTD and DDB1-DCAF1-Vpx(⌬C) SIVrcm or DDB1-DCAF1-Vpx(⌬C) SIVmnd were performed (Fig. 4, A and  B). Specifically, Hu SAMHD1 NTD amino acids were individually changed to those at the corresponding position in RCM and MND SAMHD1, i.e. S52F, E60G, V63A, E72K, and R85H (Fig. 1A). The S52F change substantially enhanced the binding of GST-Hu SAMHD1 NTD with DDB1-DCAF1-Vpx(⌬C) SIVrcm as well as with DDB1-DCAF1-Vpx(⌬C) SIVmnd (Fig. 4, A and B), whereas none of the other changes produced a similar effect. We verified that the mutation at residue 52 does not affect the structural integrity of the SAM domain by NMR and ascertained that no significant changes in the 1 H, 15 N TROSY-HSQC spectrum of the S52F mutant protein, compared with that of WT human SAMHD1 NTD, occurred (data not shown). Similarly, the 1 H, 15 N TROSY-HSQC spectra of WT and F52S MND were also essentially identical (data not shown).

Distinct Modes of SAMHD1 Recruitment to CRL4-DCAF1 by Vpx
the dissociation rate of SAMHD1 from DDB1-DCAF1, as seen by SPR (30). The increased residence time in the complex may contribute to a more efficient polyubiquitination by the CRL4 E3 ubiquitin ligase. We hypothesized that the interaction between DDB1-DCAF1-Vpx and RCM SAMHD1 may also influence the dissociation rate, and we, therefore, performed similar SPR analyses using the NTD of RCM SAMHD1 and its cognate DDB1-DCAF1-Vpx SIVrcm complex (Fig. 4C). Vpx SIVrcm substantially decreased the dissociation of the complex from the RCM SAMHD1 NTD, whereas the same virus-host protein complex dissociated from the human SAMHD1 NTD much faster (Fig. 4D). After introduction of the S52F amino acid change into human SAMHD1 NTD, a reduced dissociation rate from the DDB1-DCAF1-Vpx(⌬C) SIVrcm complex was observed (Fig. 4E). Analysis of the overall dissociation curves also suggests that the S52F amino acid change in human SAMHD1 increased the binding affinity to the virus-host protein complexes (Fig.  4F). Because the S52F mutant of human SAMHD1 was able to bind to the DDB1-DCAF1-Vpx SIVrcm complex, we also tested whether in vitro and in vivo polyubiquitination by the CRL4-DCAF1-Vpx E3 ubiquitin ligase occurs. As illustrated in Fig. 4, G and H, this is clearly the case. The importance of a Phe residue at position 52 is supported by the structure of the DCAF1-Vpx SIVmnd -SAMHD1 NTD complex. As illustrated in Fig. 4I, Phe-52 of MND SAMDH1-NTD engages in hydrophobic contacts with Phe-42 (as well as Trp-45) of Vpx SIVmnd (distances Ͻ4 Å), and identical contacts should be made with Vpx SIVrcm , as no differences in sequence in this region of Vpx SIVmnd and Vpx SIVrcm exist (Fig. 1B). Therefore, replacement by a Ser, the amino acid present at this position in human SAMHD1, will interfere with this interaction.

Discussion
One of the hallmarks revealing the presence of the evolutionary interplay between virulence factors and host restriction factors is the presence of positive selection at protein-protein interaction interfaces. This is seen by an adaptive mutation(s) in the viral proteins and localized sequence variation of the restriction factors in different host species (for reviews, see Refs. 23, 39, and 40). In particular, the co-evolution of primate SAMHD1 and Vpx of various SIVs resulted in the use of two distinctive protein-protein interfaces: NTD/Vpx and the CTD/ Vpx (27). Specifically, Vpx from SIVrcm and SIVmnd preferentially interact with the SAMHD1 NTD, whereas Vpx from SIVsm and HIV-2 requires the SAMHD1 CTD to establish antagonism, and a stable interaction only occurs when a third component, DCAF1, is present (30). The crystal structure of the DCAF1-Vpx SIVsm -SAMHD1 CTD elegantly illustrated how Vpx binding to DCAF1 creates a new molecular interface for the SAMHD1 CTD and corroborated previous biochemical findings (29). In our current work, NMR studies reveal that a large portion of the SAMHD1 NTD is affected by binding to the DCAF1-Vpx complex (Fig. 2). The crystal structure of DCAF1-Vpx SIVmnd -SAMHD1 NTD complex confirmed the NMR data. The distinct differences in the interaction of Vpx from SIVmnd and SIVsm with SAMHD1 are in stark contrast to the very similar mode observed for binding of these Vpx proteins to DCAF1 (Fig. 3, A, C, and D). Therefore, the distinct modes of SAMHD1 recruitment most likely are the result of sequence variations in Vpx, which have led to creation of unique interfaces for different regions of SAMHD1 upon DCAF1 binding. In addition to structural causes for the distinct engagement of SAMHD1 by the Vpx variants from the two species, dynamics may also play a differentiating role; real-time kinetic analyses by SPR suggest that Vpx substantially decreases the dissociation rate of SAMHD1 from the CRL4-DCAF1 E3 ubiquitin ligase, depending on whether SAMHD1 is recruited via its SAM domain (Fig. 4) or the C terminus (30).
In terms of co-evolution, it appears that the NTDs of primate SAMHD1 proteins exhibit more dramatic signatures of positive selection than the CTDs. Specifically, Gly-46 and Arg-69 are not conserved between African green monkey and MND SAMHD1s and were shown to be essential for interaction with Vpx proteins of their cognate lentiviral strains (25). Interestingly, Phe-52, which is located not far from Gly-46 and Arg-69 in the structure of the SAM domain, is affected by DCAF1-Vpx binding (Fig. 2), and the importance of a Phe residue at position 52 is clearly supported by the structure of the complex (Fig. 4I), which positions it close to the aromatic side chains of Phe-42 and Trp-45 in Vpx SIVmnd . Because the human SAMHD1 sequence possesses a serine at the corresponding position, this interaction is not possible but can be restored by the replacement of Ser-52 with Phe-52 in human SAMHD1, which renders it susceptible to down-regulation by Vpx SIVrcm (Fig. 4). These critical interface residues appear to be conserved in primate SAMHD1s, including MND and RCM. Consistent with our data, phylogenetic studies of primate SAMHD1 suggested that  C, D, and E). RU, response units. Dissociation constants were determined from two independent series of experiments. G, in vitro ubiquitination (Ubn) of full-length Hu SAMHD1 WT or S52F with CRL4-DCAF1 CA -Vpx(⌬C) SIVrcm E3 ubiquitin ligase. T7-epitope-tagged SAMHD1 proteins were incubated with E1, E2, ubiquitin, and the appropriate E3 ligases in ubiquitination buffer, as described under "Experimental Procedures." H, HEK293 cells were transiently co-transfected with DCAF1 CB (residues 1040 -1400), Hu WT or S52F SAMHD1, and Vpx SIVrcm as indicated. The levels of expressed proteins were determined by immunoblotting with appropriate antibodies. I, residue Phe-52 of MND SAMHD1 NTD (green, displayed in stick representation) is involved in hydrophobic contacts with Phe-42 and Trp-45 of Vpx SIVmnd (yellow, displayed in stick representation).
In conclusion, our studies presented here, in conjunction with several previous reports (20,(25)(26)(27)(28), provide mechanistic and structural insight at the molecular level into how Vpx proteins from different SIV lineages have adapted to sequence variations in their cellular target.
While this manuscript was in preparation a similar structure of DCAF1-Vpx SIVmnd -SAMHD1 NTD complex was reported (41).