Structural Basis of Cellular Dntp Regulation by SAMHD1 PNAS PLUS

Structural basis of cellular dNTP regulation by SAMHD1 PNAS PLUS

Xiaoyun Ji1, Chenxiang Tang1, Qi Zhao, Wei Wang, and Yong Xiong2

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520

Edited by Stephen P. Goff, Columbia University College of Physicians and Surgeons, New York, NY, and approved August 28, 2014 (received for review June 30, 2014) The sterile alpha motif and HD domain-containing protein 1 dNTPs allows it contribute to intracellular dNTP control naturally (SAMHD1), a dNTPase, prevents the infection of nondividing cells through dNTP degradation, thus influencing cell cycle progres- by retroviruses, including HIV, by depleting the cellular dNTP pool sion and DNA replication (13). available for viral reverse transcription. SAMHD1 is a major regulator SAMHD1 has an N-terminal sterile α motif (SAM) and a of cellular dNTP levels in mammalian cells. Mutations in SAMHD1 are central histidine-aspartic (HD) domain. The SAM domain is associated with chronic lymphocytic leukemia (CLL) and the autoim- a putative protein–protein and protein–nucleic acid interaction mune condition Aicardi Goutières syndrome (AGS). The dNTPase ac- module, whereas the HD domain is responsible for dNTPase and tivity of SAMHD1 can be regulated by dGTP, with which SAMHD1 viral restriction activities (1, 22–24). Structural and biochemical assembles into catalytically active tetramers. Here we present exten- studies revealed that SAMHD1 was activated by allosteric dGTP- sive biochemical and structural data that reveal an exquisite activa- induced tetramerization, which shapes the substrate-binding pocket tion mechanism of SAMHD1 via combined action of both GTP and for dNTP binding and catalysis (25). Interestingly, recent enzymatic dNTPs. We obtained 26 crystal structures of SAMHD1 in complex studies suggested that GTP is the major activator of SAMHD1 (26) with different combinations of GTP and dNTP mixtures, which depict and dNTPs substrates may also be involved in activating the en- the full spectrum of GTP/dNTP binding at the eight allosteric and four zyme (27), indicating a regulation mechanism more complex than catalytic sites of the SAMHD1 tetramer. Our data demonstrate how previously thought. SAMHD1 is activated by binding of GTP or dGTP at allosteric site 1 To understand the complete mechanism of SAMHD1 activation and a dNTP of any type at allosteric site 2. Our enzymatic assays and its potential role in cellular dNTP regulation, we carried out further reveal a robust regulatory mechanism of SAMHD1 activity, extensive structural and enzymatic studies on SAMHD1 inter-

which bares resemblance to that of the ribonuclease reductase reactions with nucleotides. Remarkably, the results reveal an exqui- BIOCHEMISTRY sponsible for cellular dNTP production. These results establish a com- sitely controlled enzymatic system, with eight allosteric binding sites plete framework for a mechanistic understanding of the important and four catalytic sites in a functional tetramer, whose activity is functions of SAMHD1 in the regulation of cellular dNTP levels, as well regulated by the combined action of GTP and all four dNTPs. The as in HIV restriction and the pathogenesis of CLL and AGS. regulation of SAMHD1 by cellular nucleotides is reminiscent of that of RNR, providing further mechanistic insight for the function HIV restriction factor | dNTP metabolism | tetramerization | of SAMHD1 in the enzymatic network of dNTP metabolism. triphosphohydrolase | allosteric regulation Results AMHD1 is a dNTPase that hydrolyzes dNTPs into deoxy- GTP Together with Any of the Four dNTPs Induces the Formation of Sribonucleosides (dNs) and triphosphates (1). It has recently Active SAMHD1 Tetramer. As GTP, instead of dGTP as previously been identified as a restriction factor that blocks infection by reported (1), was recently suggested to be responsible for a broad range of retroviruses, including HIV-1, in noncycling + SAMHD1 activation (26), we investigated whether GTP binding myeloid-lineage cells and quiescent CD4 T lymphocytes (2–7). The dNTPase activity of SAMHD1 depletes the cellular dNTP pool, Significance inhibiting the reverse transcription of the viral RNA genome (8– 10). SAMHD1 was also found to inhibit infection by certain DNA SAMHD1 is a dNTPase that depletes the cellular dNTP pool viruses including herpes simplex virus type 1 (HSV-1) and vac- to inhibit the replication of retroviruses, including HIV-1. The cinia virus in nondividing macrophages (11, 12). Apart from viral dNTPase activity of SAMHD1 also enables the enzyme to be restriction, SAMHD1 is ubiquitously expressed in both differen- a major regulator of cellular dNTP levels in mammalian cells, in tiated and undifferentiated cells of various human organs (2, 13), addition to be implicated in the pathogenesis of chronic lym- where it functions in the regulation of DNA damage signaling phocytic leukemia (CLL) and Aicardi Goutières syndrome (AGS). and proper activation of the innate immune response (14, 15). Here we present extensive structural and enzymatic data to Mutations in SAMHD1, many of which result in deficiency in the reveal how SAMHD1 is activated and regulated via the com- dNTPase activity, are associated with chronic lymphocytic leu- bined actions of GTP and all cellular dNTPs. Our work establishes kemia (CLL) (15, 16) and the autoimmune condition Aicardi- a complete spectrum of nucleotide binding and the exquisite Goutieres syndrome (AGS) (17, 18). regulatory mechanism of SAMHD1 in cellular dNTP metabolism, It has been recognized that SAMHD1 may play an important retrovirus restriction, and the pathogenesis of CLL and AGS. role in the regulation of cellular dNTP levels, which are critical to the fidelity of DNA synthesis and the stability of the genome (13). Author contributions: X.J., C.T., and Y.X. designed research; X.J., C.T., and W.W. per- Abnormal size and/or the imbalance of the dNTP pool may acti- formed research; X.J., C.T., Q.Z., and Y.X. analyzed data; and X.J., C.T., and Y.X. wrote vate the S-phase checkpoint for cell cycle arrest (19, 20). In the paper. mammalian cells, the concentration of cellular dNTPs is tightly The authors declare no conflict of interest. regulated and maintained during the cell cycle, notably by ribo- This article is a PNAS Direct Submission. nucleotide reductase (RNR) during dNTP synthesis. RNR increases Data deposition: The atomic coordinates and structure factors have been deposited in the the dNTP pool by converting ribonucleoside diphosphates Protein Data Bank, www.pdb.org (PDB ID codes 4TNP, 4TNQ, 4TNR, 4TNX, 4TNY, 4TNZ, (NDPs) into dNDPs in a tightly controlled cell cycle-dependent 4TO0, 4TO1, 4TO2, 4TO3, 4TO4, 4TO5, and 4TO6). manner (21). The activity of RNR is elegantly regulated by the 1X.J. and C.T. contributed equally to this work. binding of ATP, dATP, dTTP, and dGTP at two allosteric sites, 2To whom correspondence should be addressed. Email: [email protected]. which results in balancing of the production of all four dNDPs in the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. catalytic site of the enzyme. The ability of SAMHD1 to hydrolyze 1073/pnas.1412289111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1412289111 PNAS Early Edition | 1of10 Downloaded by guest on September 25, 2021 Fig. 1. GTP together with any dNTP, but not GTP alone, efficiently induces tetramerization of SAMHD1. (A) Size exclusion chromatograms of SAMHD1c-RN incubated with dGTP or GTP. Purified samples of SAMHD1c-RN (2 mg/mL, 200 μL) mixed with a final concentration of 4 mM dGTP or 4 mM GTP were applied to a Superdex 200 10/300 GL column. (B) Sedimentation velocity AUC results for SAMHD1c (1 mg/mL) with GTP (100 μM) or dGTP (100 μM). Little tetramer was formed with GTP alone. (C) Sedimentation velocity AUC results for SAMHD1c (1 mg/mL) with GTP (100 μM) and dNTP (100 μM) of any type.

alone can lead to the active tetrameric form of SAMHD1. Our the enzyme. The results show that SAMHD1c-RN incubated with results show that incubation of SAMHD1 with only GTP does GTP eluted from the column at a volume corresponding to a dimer, not yield stable tetramers (Fig. 1). We used the inactive mutant in contrast to the corresponding tetramer position for SAMHD1c- of human SAMHD1 catalytic core SAMHD1c-RN (residues 113– RN with dGTP (Fig. 1A). For independent verification, we mea- 626 with mutations of H206R/D207N), which has been shown suredthesedimentationrateofSAMHD1c-RNwith100μMdGTP previously to have identical nucleotide positioning and oligo- or GTP using sedimentation velocity analytical ultracentrifugation merization properties as the active catalytic core (25). We first (SV-AUC). Indeed, SAMHD1c-RN with dGTP sedimented as a incubated SAMHD1c-RN with 4 mM dGTP or GTP and used tetramer as reported previously (28), whereas little tetramer was size exclusion chromatography to examine the oligomeric state of detected in the sedimentation profile for SAMHD1c-RN with

Fig. 2. Crystal structures of SAMHD1 tetramer in complex with GTP and a dNTP of any type. (A) Structure of SAMHD1 tetramer bound to nucleotides (Left). The four subunits are shown as ribbon or surface presentation, colored in light blue, pale cyan, salmon, and wheat, respectively. Nucleotides are shown as sticks. The representative allosteric and catalytic nucleotides in one subunit (salmon) were highlighted with green meshes. (Right) The schematic of the 2+ tetramer. (B–D) Unbiased Fo-Fc difference electron density (σ = 3.0) of the nucleotides bound to the SAMHD1 tetramer. (B) The allosteric site dGTP-Mg -dGTP + (PDB ID code 4BZB). (C) The four scenarios of allosteric site GTP-Mg2 -dNTPs. (D) The dNTP substrates at the catalytic site.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1412289111 Ji et al. Downloaded by guest on September 25, 2021 GTP (Fig. 1B). These experiments demonstrate that, unlike dGTP, units in the SAMHD1 tetramer adopt the same conformation PNAS PLUS GTP does not efficiently induce stable SAMHD1 tetramers. (RMSD < 0.25 Å), each containing two allosteric nucleotide- Because SAMHD1 has two allosteric nucleotide-binding sites binding sites and a catalytic nucleotide-binding site. The two for each of the four subunits in the tetramer, it is possible that GTP nucleotides at the allosteric sites are closely juxtaposed in an ar- + and dNTP act in a cooperative manner to induce the tetramers and chitecture that had been observed before, with a Mg2 ion bridging activate the enzyme. In fact, in the work that reported GTP as the their phosphate groups (Figs. 2 and 3A). Interestingly, one of the primary activator, dNTP substrates were always present, together two allosteric sites (designated as Allo-site 1) can only be occupied with GTP, in the SAMHD1 activity assays (26). To test the hy- by GTP or dGTP, whereas the other (designated as Allo-site 2) can pothesis that a combination of GTP and dNTP induces tetrame- bind all dNTPs. The two bound nucleotides have a GTP/dGTP– + rization of SAMHD1, we mixed GTP and a dNTP (100 μM) with Mg2 –dNTP configuration (Figs. 2 and 3A), which includes the + SAMHD1c-RN and analyzed its oligomer state by SV-AUC. In- previous observed dGTP–Mg2 –dGTP configuration (25, 29). deed, the presence of GTP and a dNTP caused SAMHD1 to sedi- ment at a position corresponding to a tetramer (Fig. 1C). These Allo-Site 1 Strictly Selects Guanine Nucleotides. Guanine nucleo- results, together with the previous observations that dGTP, but not tides, GTP or dGTP, are exclusively selected at Allo-site 1, any other dNTP, alone can induce tetramerization, suggest that the where a network of five specific hydrogen bonds are formed formation of the active SAMHD1 tetramer requires a guanine nu- between the base edge of guanine and the surrounding residues of cleotide (GTP or dGTP) together with another dNTP of any type. D137, Q142, and R145. Nucleotides of other base identity, such as dATP, dTTP, or dCTP, cannot bind to Allo-site 1 even with the Crystal Structures of Tetrameric SAMHD1 in Complex with GTP and high concentrations (up to 20 mM) as used in the cocrystallization adNTPofAnyType.To understand the molecular basis of SAMHD1 experiments. Superposition of the GTP- or dGTP-bound SAMHD1 activation by GTP and dNTPs, we cocrystallized SAMHD1c-RN at Allo-site 1 showed only minor structural changes for the protein with GTP and each of the four dNTPs at resolutions ranging from on binding of the two nucleotides, which adopt nearly identical 2.2 to 2.9 Å. The structures were solved by molecular replacement conformations including sugar puckering (Fig. 3 A and B). The only using the dGTP-complex of tetrameric SAMHD1c-RN [Protein difference between the two structures is a small shift (∼0.4 Å) of Data Bank (PDB) ID code 4BZB] (25), with the bound dGTP residues adjacent to the GTP/dGTP-binding Allo-site 1 (residues excluded, as a search model. Unbiased difference electron density 114–121). The observed shift is significant considering the data and (F -F ) of high quality clearly revealed the identity of the bound model quality (30). This shift enables an extra hydrogen bond to be

o c BIOCHEMISTRY nucleotides and allowed for successful model building (Fig. 2). formed between the 2′-OH group of GTP and the main chain ox- Data collection and refinement statistics are listed in Table 1 and ygen of V117. This additional interaction suggests a modestly higher Table S1. affinity for GTP at this site. To verify this prediction, we cocrystal- Consistent with our AUC results, SAMHD1c-RN in complex lized SAMHD1c-RN with various concentrations of GTP and with GTP and various dNTPs crystallized as tetramers with vir- dGTP together and inspected the nucleotide species at Allo-site 1 in tually identical protein structures (RMSD 0.66–0.85 Å) to that the resulting crystal structures. As expected, whenever equal or of SAMHD1c tetramer observed previously (25). The four sub- higher concentration of GTP was mixed with dGTP, GTP was found

Table 1. Summary of cocrystallization and ligand binding data

Data Cocrystallization nucleotides concentration (mM) ratio Resolution (Å) Rwork/Rfree Allo-site 1 Allo-site 2 Catalytic site

1 GTP:dATP 4:1 2.6 0.22/0.24 GTP dATP dATP* 2 GTP:dATP 1:4 2.9 0.19/0.23 GTP dATP dATP* 3 GTP:dATP 1:10 2.8 0.22/0.24 GTP dATP dATP* 4 GTP:dGTP 4:0.4 2.3 0.17/0.21 GTP dGTP dGTP* 5 GTP:dGTP 2:2 2.6 0.21/0.25 GTP dGTP dGTP* 6 GTP:dTTP 4:1 2.4 0.19/0.24 GTP dTTP dTTP* 7 GTP:dTTP 1:4 2.4 0.20/0.24 GTP dTTP dTTP 8 GTP:dTTP 1:20 2.6 0.16/0.20 GTP dTTP dTTP 9 GTP:dCTP 1:4 2.4 0.18/0.20 GTP dCTP dCTP 10 GTP:dATP:dGTP 1:4:4 2.6 0.21/0.24 dGTP dATP dGTP 11 GTP:dATP:dTTP 1:4:4 2.4 0.21/0.24 GTP dATP dTTP 12 GTP:dATP:dCTP 1:4:4 2.8 0.21/0.23 GTP dATP dCTP 13 GTP:dATP:dCTP 1:1:4 1.9 0.19/0.21 GTP dATP dCTP 14 GTP:dATP:dCTP 1:0.1:5 2.3 0.19/0.22 GTP dATP dCTP 15 GTP:dATP:dCTP 1:0.1:10 2.6 0.22/0.24 GTP 2dATP:2dCTP dCTP 16 GTP:dATP:dCTP 1:0.1:20 2.0 0.20/0.22 GTP dCTP dCTP 17 GTP:dATP:dCTP 1:20:0.4 2.9 0.19/0.22 GTP dATP † † 18 GTP:dATP:dCTP 1:20:0.1 2.5 0.17/0.21 GTP dATP 19 GTP:dGTP:dTTP 1:4:4 2.3 0.19/0.22 dGTP dGTP 3dGTP:1dTTP 20 GTP:dGTP:dCTP 1:4:4 2.1 0.16/0.21 dGTP dGTP dCTP 21 GTP:dGTP:dCTP 1:1:10 2.1 0.20/0.23 GTP dGTP dCTP 22 GTP:dTTP:dCTP 1:4:4 2.8 0.23/0.26 GTP dTTP dCTP 23 GTP:dTTP:dCTP 1:1:10 2.9 0.21/0.24 GTP dTTP dCTP 24 GTP:dGTP:dATP:dTTP 1:4:4:4 2.4 0.22/0.24 dGTP dATP 3dTTP:1dGTP 25 GTP:dATP:dTTP:dCTP 1:4:4:4 2.1 0.17/0.20 GTP dATP dCTP 26 GTP:dGTP:dCTP:dTTP 1:4:4:4 2.4 0.21/0.24 dGTP dGTP dCTP

*The base portion of the nucleotide is not fully observed. †Types of nucleotide could not be determined.

Ji et al. PNAS Early Edition | 3of10 Downloaded by guest on September 25, 2021 + + + Fig. 3. The structures of the GTP-Mg2 -dNTPs at the allosteric sites. (A) Overlay of the structures of dGTP-Mg2 -dGTP and four dGTP-Mg2 -dNTPs. Nucleotides are + shown as sticks and Mg2 as spheres. Allo-site 1 nucleotides are colored in orange. Allo-site 2 nucleotides are colored individually according to dNTP types: dATP in yellow, dGTP in green, dTTP in magenta, and dCTP in cyan. (B) Superposition of Allo-site 1 structures of dGTP- or GTP-bound SAMHD1. SAMHD1 structures (pink: dGTP-bound, cyan: GTP-bound) are shown as ribbon with side chains of labeled residues shown as sticks. GTP and dGTP are shown in orange and pink, respectively. (Inset) Extra hydrogen bond (dashed line) between the 2’-hydroxyl group of GTP and main chain oxygen of V117. (C) Superposition of the structures of the four different dNTPs at Allo-site 2 of SAMHD1. dNTPs and labeled protein side chains are shown as sticks. The dATP-bound structure (yellow) is shown thicker than those of the other three. R333 stabilizes the bound dNTP through stacking interaction. The rigid side chain of F157 would cause steric clash (marked by a red X)withthe 2′-OH group of an NTP. (D) The side chains of the surrounding residues adopt different conformations to adapt to the four different dNTP bases bound at Allo-site 2.

to occupy Allo-site 1. A structure with dGTP bound to Allo-site 1 unable to form the allosteric nucleotide pair to induce the tet- was obtained with a dGTP/GTP ratio of 4. Considering that the ramerization of SAMHD1 (Fig. 1). In fact, we could not obtain physiological concentration of GTP is 1,000-fold higher than dGTP any crystals of SAMHD1 with GTP alone or together with any (31), we conclude that GTP occupies Allo-site 1 exclusively in cells. other NTPs. In contrast, SAMHD1 crystals are readily grown in the presence of GTP and a dNTP, where different dNTPs can Allo-Site2CanAccommodateadNTPofAnyType,butNotNTPs. C Allo-site 2 precludes the binding of NTPs but is promiscuous for bind to Allo-site 2 with almost identical conformations (Fig. 3 ). any dNTP. Cocrystallization of SAMHD1 with GTP and any A flexible interaction network accommodates the binding of the dNTP, including with 10-fold excess of GTP over dGTP, did not four dNTPs, with the side chains of N119, D330, N358, and R372 yield structures with GTP bound to Allo-site 2 (Table 1). GTP is changing conformations to maintain two hydrogen bonds with likely excluded because of the steric clash between its 2-OH′ each of the different bases (Fig. 3D). group and the aromatic side chain of F157 in the tight binding Base-stacking interactions also stabilize the nucleotides at the pocket (25) (Fig. 3C). This exclusion explains why GTP alone is allosteric sites and can potentially lead to affinity variations for

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1412289111 Ji et al. Downloaded by guest on September 25, 2021 PNAS PLUS BIOCHEMISTRY

Fig. 4. The structures of the dNTP substrates at the catalytic site. (A) Superposition of the structures of different dNTPs bound to the catalytic site of SAMHD1. The color scheme of the dNTPs is the same as in Fig. 3. The structure of the SAMHD1-dGTP complex is in semitransparent ribbon presentation (wheat). dNTPs and critical side chains were shown as sticks. (B) dATP binds weakly to the substrate-binding pocket (surface) with no clear conformation observed for the adenine base (semitransparent sticks). (C) The substrate-binding pockets (surface) and the bound dCTP, dTTP, and dGTP (sticks). Extensive water molecules (red spheres) at the dNTP base edges bridge the nonspecific interactions with the substrate-binding pocket. Perpendicular views of the base and water surfaces are shown in the lower panels. The pyrimidine bases (dCTP and dTTP) appear to have more extensive water networks than that of the purine base of dGTP.

dNTP binding at Allo-site 2. In particular, the guanidinium group networks than those of purine dNTPs (Fig. 4 B and C). These of R333 stacks with the base of the dNTP at Allo-site 2. Mutation extensive water networks could potentially favor dCTP/dTTP of R333A has been shown to affect SAMHD1 tetramerization and binding at the catalytic site. We did not observe full occupancy abolish its function (25). The stacking interaction suggests a bind- of dATP at the catalytic site (Fig. 2D), even with the highest ing preference where the larger purine dNTPs are potentially concentration (10 mM) among all dNTPs used for cocrystalliza- favored at Allo-site 2. Interestingly, only when dATP binds to tion. This observation suggests that dATP may have the weakest Allo-site 2 does the side chain of E355 move to further stabilize binding affinity at the catalytic site. It should be noted that the RN R333 by forming additional salt bridges (Fig. 3C). This local mutation of SAMHD1 does not alter the positioning of the dNTP environment may subsequently increase the stability of the substrate in the native conformation (25). It is possible that the stacked dATP and result in a higher affinity for dATP over the overall binding affinities are changed due to the mutation affecting other dNTPs at Allo-site 2. the strength of interaction with the triphosphate group. How- ever, the relative binding preference/selectivity to various dNTPs The Catalytic Site Accommodates Any dNTPs. We obtained crystal should be maintained, as this is dictated by SAMHD1 interaction structures of SAMHD1c-RN with each different dNTP bound to with the bases and is not affected by the mutation. the catalytic site. The nucleotide-binding pocket and the bound dNTP adopt nearly identical conformations as seen in the pre- Relative Binding Affinities for Different dNTPs. We used a structure- viously reported SAMHD1-dGTP structure (25) (Fig. 4A). There based approach to assess the binding affinity of dNTPs at the allo- is no specific contact between the enzyme and the base edge of steric and catalytic sites, using an additional 18 crystal structures any dNTPs, consistent with the ability of SAMHD1 to hydrolyze of SAMHD1 cocrystallized with various nucleotide mixtures. The all four dNTPs. Instead water molecules mediate interactions structure-based approach was necessary because of the complexity between the base edge of the dNTPs and the enzyme non- of the large number (12) of nucleotide-binding sites in the specifically. Interestingly, it appears that dNTPs with pyrimidine SAMHD1 tetramer. The large number of binding sites, together bases (dCTP and dTTP) have more extensive bridging-water with the monomer/dimer to tetramer transitions induced by

Ji et al. PNAS Early Edition | 5of10 Downloaded by guest on September 25, 2021 GTP/dNTP binding, prohibits the analysis by standard solution of the binding preference is dCTP > dGTP/dTTP > dATP. We biophysical techniques. We set up a series of simultaneous coc- observed uniform binding of a dNTP at the four equivalent Allo- rystallization trials for SAMHD1c-RN in the presence of 1 mM sites 2 or catalytic sites in a SAMHD1 tetramer in most of the GTP and different combinations of dNTPs. In each cocrystal- cases. This observation indicates that under the experimental lization experiment, a mixture of dNTPs was used for either conditions, such as when equal concentrations of all four dNTPs pairwise or three-dNTP binding competition at various ratios. are presented to the enzyme, the affinity differences between the All of the crystals used in the analysis diffracted to resolutions nucleotides are large enough to ensure binding of the same dNTP between 2.0 and 2.9 Å, which yielded clear unbiased electron at all equivalent sites. Interestingly, when the ratio of the nucleo- density for the bound nucleotides (Fig. S1). The data and re- tides was varied, we observed mixed binding of different dNTPs at finement statistics are summarized in Table 1 and Table S1. equivalent sites in the tetramer in three cases (Table 1). This finding Our comprehensive structure-based binding analysis shows demonstrates that SAMHD1 can tetramerize and be activated by that GTP always occupy Allo-site 1 under physiological con- binding of a mixture of dNTPs and can potentially hydrolyze dif- ditions (as discussed above), and interestingly, the order of ferent substrates simultaneously within the same tetramer complex. preference for dNTPs at Allo-site 2 tends to be the opposite of that at the catalytic site. As anticipated from the structural GTP and Any of the Four dNTPs Can Allosterically Activate SAMHD1. analysis, purine nucleotides showed higher affinity to bind Allo- As GTP together with any dNTP can promote the formation of site 2 than pyrimidine nucleotides, with the order of dATP > SAMHD1 tetramer required for the dNTPase activity, we next dGTP > dTTP > dCTP. Notably, dATP is the favored ligand at investigated whether GTP and dNTP act in a cooperative man- Allo-site 2 and cannot be competed off even with a 100-fold ner to activate SAMHD1. WT SAMHD1c was incubated with higher concentration of dCTP (Table 1). However, dATP dis- GTP and a dNTP at various equal concentrations (0–800 μM), played the weakest binding at the catalytic site, where the order and the reaction product dN was monitored by HPLC. This

Fig. 5. Steady state kinetics for SAMHD1 dNTPase activity. All experiments were performed in triplicate. Schematic of the experimental setup for each assay is shown above the plots. (A) Single-dNTP substrate dNTPase assays. The dNTPase activity of SAMHD1c (0.5 μM) was assayed with indicated concentrations of dNTP, together with the same concentrations of GTP, for 5–15 min. The amount of dN product generated in the reactions was quantified by HPLC. (B)dNTPaseassays with a mixture of all four dNTP substrates. Experiments were performed with a mixture of four dNTPs (500 μM each), together with SAMHD1c (0.5 μM) and GTP (500 μM). The linear initial rates of dN production were plotted. (C) Comparison of the dNTP hydrolysis rates in the single-substrate (black columns) and the four- substrate (white columns) assays at the dNTP(s) concentration of 500 μM. Each rate was normalized to that at the same concentration ratio of enzyme to total dNTPs. (D) dNTPase assays with preassembled SAMHD1 tetramers. SAMHD1c tetramers were preassembled by incubation with GTP and a particular Allo-site 2

dN2TP as indicated. After 100-fold dilution with reaction buffers containing a specific dN1TP substrate, the product was quantified and plotted as indicated. (E) dNTPase assays with pair-wise dNTP substrates. SAMHD1c (0.5 μM) activity assay were performed with GTP (500 μM) and a pair of dNTPs (500 μMeach).The dN1-dN2 production rates of dNs were quantified and kapp values were calculated as described in Materials and Methods. The rates are displayed as indicated. (F) dN2-dN1 Validation of the calculated kapp values by the predicted effective enzyme utilization in the independent dNTPase assays with 3-dNTP or 4-dNTP mixtures. The dN2-dN1 kapp values obtained in E lead to a calculated total effective enzyme utilization of 100% in each experiment, indicating the rates were correctly obtained.

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1412289111 Ji et al. Downloaded by guest on September 25, 2021 experimental design ensured that Allo-site 1 was occupied by GTP, problem of the preassembled SAMHD1 tetramers. This assay is PNAS PLUS and a specific dNTP was present in both Allo-site 2 and the based on measurements of individual dN products within dif- catalytic site. The results confirmed that in the presence of GTP ferent pairwise combinations of dNTPs that were presented to and dNTP, SAMHD1 was indeed activated to hydrolyze dNTPs SAMHD1 with GTP (Table S2). A key to this approach of rate rapidly. The apparent turnover rates vary up to twofold for dif- analysis is to delineate the effective concentration of the SAMHD1 ferent dNTP substrates in the order of dATP > dTTP > dCTP > enzyme, with a particular dNTP cofactor at Allo-site 2, used by each dGTP (Fig. 5A). The reaction profiles were fitted to a sigmoidal dNTP substrate in the mixture, [SAMHD1dN1-dN2]. The analysis kinetic function, which resulted in various degrees of sigmoidal was made possible by our knowledge of the hydrolysis rates dNi-dNi shaped curves expected from cooperativity effect due to allosteric determined in the single-substrate experiments (kapp )and binding. The dGTP reaction showed the lowest cooperativity, which the relative ranking of dNTP affinities at Allo-site 2 obtained is consistent with that observed previously (27). In contrast, the from our structure based affinity assays. For example, when dCTP reaction profile showed the most pronounced sigmoidal be- equal concentrations of dATP and dCTP are mixed with havior of activation. This reaction profile agrees with our crystal- SAMHD1/GTP, dATP will occupy Allo-site 2 because of its lographic result that dCTP has the lowest binding affinity at Allo- much higher affinity (Table 1). We can then calculate site 2 and thus has the least capacity to induce tetramerization. [SAMHD1dA-dA] by comparing the dA production rate measured in the dATP/dCTP mixture experiment with that GTP and All dNTPs Together Control the Rates of Substrate Hydrolysis. obtained with dATP alone. This calculation in turn gives the To examine how GTP and all four dNTPs together control remaining fraction of SAMHD1 used by the dCTP substrate, SAMHD1 activity, as is the situation in vivo, we mixed an equal [SAMHD1dC-dA]. k dC-dA can finally be calculated by dividing μ app amount of all four dNTPs (500 M each) with SAMHD1c in the the measured production rate of dC by [SAMHD1dC-dA]. μ presence of 500 M GTP and analyzed the reaction products (Fig. We obtained 10 possible kdN1-dN2 values from experiments B app 5 ). The results show that in the presence of all four dNTP sub- performed with all combinations of dNTP pairs at equal con- > > strates, the hydrolysis rates are in the order of dGTP dCTP centrations (Fig. 5E and Table S2), which reflect the cellular > dTTP dATP, which is the opposite of that obtained in the single condition of balanced dNTP pools in the cell. Under these con- > > > dNTP substrate experiments (dATP dTTP dCTP dGTP) ditions, some turnover events are not preferred as our structure- C (Fig. 5 ). The order of rates obtained with the mixture of all based affinity assays showed that the dNTP with higher affinity dNTPs agrees with the previous observations in vitro with Allo-site dominates Allo-site 2. For example, little dCTP will occupy Allo- 1 of SAMHD1 occupied by dGTP (1, 24, 28) and the siRNA si- site 2 in the presence of equal concentration of dATP, i.e., only BIOCHEMISTRY lencing experiment in vivo (13). The rate changes between the dA-dA dC-dA dA-dC kapp or kapp , but not kapp , is relevant and measurable. In single dNTP and the dNTP mixture assays may reflect potential fact, in our experiment with a mixture of all four dNTPs described cross-talk between the allosteric sites and the catalytic site, i.e., above, dATP (highest affinity) is most likely incorporated into the different apparent turnover rates for the same dNTP substrate majority of Allo-site 2 in each enzyme, which then acts on four when Allo-site 2 was occupied by different dNTP cofactors. The dN-dA dNTP substrates with individual apparent turnover rates of kapp overall rate change may also be a result of the competition of (Fig. 5C). The results from the dNTP-pair experiments again various dNTPs for the same catalytic sites, even though the in- show the hydrolysis rates of a dNTP do not differ significantly dividual rates do not differ by very much. with different allosteric dNTPs (Fig. 5E), confirming the observation made from the preassembled SAMHD1. Different Allosteric dNTP Cofactors Activate SAMHD1 to a Similar To further verify our experimental design and calculations, we Level. We used multiple approaches to investigate whether dif- also performed activity assays with all combinations of three dNTPs ferent allosteric dNTPs affect the catalysis rate for any given as substrates. The procedure is similar to that described for the substrates. We analyzed the apparent turnover rate of each dN1-dN2 pairwise dNTP experiments with a variation in analysis. In this case dNTP with various dNTP cofactors at Allo-site 2, kapp (dN1: the measured dN production rates, together with the kapp values substrate dNTP; dN2: Allo-site 2 dNTP). Our first approach uses obtained earlier, were used to predict the amount of effective SAMHD1 tetramers preassembled with a specific dNTP and SAMHD1 used by each dNTP substrate in the 3-dNTP mixtures. measures the rate of hydrolysis of other dNTPs. This approach Only when the kapp values were accurately obtained in the 2-dNTP is possible because our previous biochemical data indicate that experiments can we use them to predict the correct effective enzyme SAMHD1 tetramers are stable and may perform multiple cata- – utilization by different substrates in the 3-dNTP mixtures, which lytic cycles without having to go through the dimer tetramer should sum up to 100% in each experiment. We also performed transition (28). This approach is also supported by the recent the same analysis for the experiment with the mixture of all four report that the activated state of SAMHD1 is long-lived even dNTPs. Indeed, the total effective enzyme used by all dNTPs in when dNTP levels are subsequently reduced to very low levels each experiment converged to ∼100%, which validates our calcu- (27). We preincubated SAMHD1c with GTP (500 μM) and a μ lations and supports the result that the apparent turnover rates for a specific dNTP (dN2, 500 M) to form the active tetramer. The given dNTP substrate are basically unaltered when different dNTP solution was subsequently diluted 100-fold rapidly by adding cofactors are bound at the allosteric site of SAMHD1 (Fig. 5F). the assay buffer containing the desired dNTP substrate (dN1, μ These experiments demonstrate that SAMHD1 has an overall 500 M) to initiate the reaction. The results show that the ap- regulation mechanism where any allosteric dNTP cofactor likely parent turnover rate for a substrate barely changed when Allo-site 2 D activates the enzyme to a similar extent. The final reaction rates for was occupied by different dNTPs (Fig. 5 ). These experiments the dNTP substrates are then controlled by their competition for demonstrate that different allosteric dNTPs activate SAMHD1 the catalytic sites. This property is exemplified in the case of dATP, to a similar level. A potential caveat of this assay is that the dA-dA where there is only a single, relatively large rate kapp in experi- preassembled SAMHD1 tetramer is not stable and allows for ments with either dATP alone or the mixture of all four dNTPs. the exchange of substrate dNTPs into Allo-site 2. Consequently, However, the rate of dA production in the 4-dNTP mixture kdN1-dN2 kdN1-dN1 the measured rate would be a mixture of app and app dropped to below half of that of dATP alone, which can be dN1-dN1 and may even be dominated by kapp due to the 100-fold higher explained by the weak ability of dATP to compete for SAMHD1 concentration of dN1 vs. the concentration of dN2. active sites. This explanation is consistent with our structure- We used a second approach to confirm the measured apparent based affinity analysis, where dATP has the lowest affinity at the dN1-dN2 turnover rates kapp while avoiding the potential dissociation catalytic site. Small variations of kapp values are observed for the

Ji et al. PNAS Early Edition | 7of10 Downloaded by guest on September 25, 2021 same dNTP substrate with SAMHD1 bound with different dNTP cofactors, which can be a result of a subtle regulation of SAMHD1 activity by different dNTPs at Allo-site 2. Our steady-state kinetic analysis offers an initial evaluation of the overall rate-limiting step of the complex reactions of dNTP hydrolysis by SAMHD1. More comprehensive analysis is required to dissect the detailed kinetic reaction pathway. Discussion SAMHD1 has been proposed to play an important role in the control of cellular dNTP pools, in addition to its functions in preventing viral infection and autoimmunity (13, 32). A central question with respect to these SAMHD1 functions is how the dNTPase activity of the enzyme is regulated by dNTP metabolism. It has been demonstrated previously that SAMHD1 is activated by allosteric dGTP- and/or GTP-induced tetramerization. Although the cellular dGTP concentration may provide a conve- nient signal for activity control, sensing the level of a single nucleotide only (dGTP) does not appear to be a robust regulatory mechanism. Based on SAMHD1 activity on dGTP and dUTP, it has been recently proposed that GTP and dNTP substrates activate SAMHD1 together (27). However, fundamental questions about whether and how GTP collaborates with each of the four dNTPs to active SAMHD1 and the regulation of SAMHD1 activity by different cellular dNTPs have not been elucidated. The study presented herein establishes a comprehensive Fig. 6. SAMHD1 and RNR regulate cellular dNTP pool together. RNR and structural and biochemical framework for understanding the SAMHD1 are responsible for the production and degradation of dNTPs, re- important functions of SAMHD1 in cellular dNTP regulation. spectively. Both of them have three specific nucleotides binding sites as indicated, with the types of the nucleotides that can occupy the sites marked Our crystal structures and enzymatic assays illustrate a robust on the side. The concentrations of cellular dNTPs are sensed and regulated regulatory mechanism by which GTP and all four dNTPs act to- by both of the enzymes. dATP is produced last by RNR and degraded last gether to control the activity of SAMHD1. Although both GTP by SAMHD1. and dGTP can occupy Allo-site 1, GTP should be the primary activator given its intrinsic higher affinity to the site and its much higher cellular concentration. It appears that all four dNTPs can activity at a specific catalytic site. Second, the activity of both activate SAMHD1 to a similar level, making the enzyme a robust, enzymes is finely regulated by binding of nucleotides at the re- general sensor of cellular dNTP pools. Interestingly, under con- spective allosteric sites, which influences the conformations of the ditions of a balanced cellular pool where all dNTPs are at a similar catalytic sites to either activate or inactivate the enzymes. Third, concentrations, it is most likely that dATP occupies Allo-site 2 due in each case Allo-site 1 is nucleotide base specific and Allo-site 2 to its highest affinity for this site (Table 1). Therefore, the most has binding promiscuity toward multiple dNTPs. Specific NTP/ probable activator configuration at the two juxtaposed allosteric dNTP binding at the Allo-site 1 of either enzyme controls the re- + sitesisGTP–Mg2 –dATP, which should occur under normal cel- spective catalytic activity, although in different ways. Human RNR is lular conditions in nondividing cells and the G1 phase of dividing activated as a α2β2 heterotetramer when ATP is bound to Allo-site 1, cells. Nonetheless, the ability of SAMHD1 to sense all dNTPs whereas binding of dATP at the same site inhibits the activity. In enables a general feedback system to achieve a balanced cellular contrast, the SAMHD1 homotetramer is activated by binding of dNTP pool in uncommon or extreme conditions where cellular either dGTP or GTP at the Allo-site 1. Another major difference dNTPs are unbalanced, such as during malfunction in cellular dNTP between the two enzymes is that ATP and dNTPs bound to the al- production or during the invasion of certain dsDNA viruses that losteric sites of RNR control its activity on nucleoside diphosphates manipulate the dNTP pool to favor viral reproduction (12). (rNDPs) substrates bound to the catalytic sites, whereas dNTPs are Our study provides a structural basis and complementary anal- used as both allosteric activators and substrates for SAMHD1. ysis to the reported study on dGTP/dUTP catalysis by SAMHD1 SAMHD1 and RNR may have evolved to use cellular dATP (27). During the preparation of this manuscript, Hansen et al. (27) levels as an overall control switch for their activities, albeit in reported the combined activation of SAMHD1 tetramer by GTP opposite ways as dictated by the reciprocal function of the two and dUTP. Based on enzymatic activity assays for dUTP/dGTP enzymes. dATP is the last to be synthesized by RNR in a syn- hydrolysis and structural modeling, the report predicted that any thetic cycle and is used as a feed-back inhibitor for switching off type of dNTP could bind to Allo-site 2 and activate SAMHD1 in the production of cellular dNTPs by RNR (33). In contrast, our a similar way. Our crystal structures confirm the proposal and data show that dATP has the highest affinity for the allosteric provide a structural explanation to the reported experimental data. site of SAMHD1, enabling it to be the most sensitive activator Furthermore, our kinetic analysis extends to all cellular dNTPs, for dNTP degradation. In addition, dATP has the weakest af- including analysis of double- and triple-nucleotide combinations, finity for the catalytic site of SAMHD1 and has the slowest hy- which result in reaction profiles and range of reaction rates con- drolysis rate when all dNTPs are present (Fig. 5B); therefore, it is sistent with the published data from single-nucleotide experiments likely the last dNTP to be depleted in the cell. In summary, the on dUTP or dGTP. These complementary results further illumi- coordinated uses of dATP facilitate the coordination of the activ- nate the dNTPase activity of SAMHD1. ities of both RNA and SAMHD1. The last nucleotide made in the Striking connections exist between the enzymatic properties of dNTP production pipeline is dATP and a high level of which will SAMHD1 and RNR (Fig. 6), which are responsible for cellular shut down the production enzyme RNR. A high level of dATP dNTP degradation and production, respectively. First, both of simultaneously activates the degradation enzyme SAMHD1, whose the enzymes are active oligomers with two types of allosteric sites dNTPase activity only stops after all dNTPs are brought to low that sense the cellular nucleotide concentrations and control the steady state levels, with dATP being the last to be degraded (Fig. 6).

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1412289111 Ji et al. Downloaded by guest on September 25, 2021 Maintaining a balanced cellular dNTP pool at an appropriate mixed at the indicated concentrations and incubated at 25 °C. Aliquots of re- PNAS PLUS level is of paramount importance for cell cycle progression, ge- action samples were collected at various times points with the reaction termi- nome integrity, proper immune activation, and viral restriction of nated by 5× dilution into ice cold buffer containing 10 mM EDTA, followed by × viruses (19, 34, 35). The opposite, cell cycle-dependent manner spinning through an Amicon Ultra 0.5 mL 10 kDa filter (Millipore) at 16000 g for 20 min. Deproteinized samples were analyzed by HPLC using a Synergi C18 of regulation of RNR and SAMHD1 expression is the major con- Column 150 × 4.6 mm (Phenomenex). The column was pre-equilibrated in 20 mM trol mechanism for maintaining the desired dNTP concentrations ammonium acetate, pH 4.5 (buffer A). Injected samples were eluted with in the dividing cells. The expression of SAMHD1 is minimized, a linear methanol gradient over 14 min at a flow rate of 1 mL/min. The yields whereas the production of RNR is maximized during S-phase were quantified by integration of the calibrated UV absorption peak at 260 nm. when large amount of dNTPs are essential for optimal rates of In the single-dNTP substrate assay, the initial rates of product formation DNA replication (13). The work presented here provides a struc- catalyzed by 0.5 μM SAMHD1c were measured up to no more than 20% tural and enzymatic basis for understanding how SAMHD1 joins completion and plotted against the substrate concentration of each dNTP. RNR for dNTP regulation in nondividing cells and G1 phase The reaction profiles were fitted to a sigmoidal kinetic function (Eq.1), of dividing cells, where the coordinated actions between the two V × ½dNTPh enzymes helps to keep the cellular dNTP pool low and balanced. v = max [1] Kh + ½dNTPh This insight also forms a foundation for deciphering the mechanisms half of SAMHD1 functions in HIV restriction and the pathogenesis of chronic lymphocytic leukemia and Aicardi Goutières syndrome. where Vmax is the maximum dNTP production rate, Khalf is the concentration of substrate that produces a half-maximal enzyme velocity and Materials and Methods h is the Hill coefficient. In the preassembled tetramer assay, samples containing of purified Protein Expression and Purification. N-terminal 6× His-tagged SAMHD1c SAMHD1c (50 μM) were preincubated with GTP (500 μM) and a particular (residues 113–626) and its RN mutant (H206R D207N) were expressed in dNTP (500 μM) for 1 min before diluted 100-fold rapidly with the assay Escherichia coli and purified using nickel-nitrilotriacetic acid (Ni-NTA) affinity buffer containing 500 μM a desired substrate dNTP to initiate reactions, and and size-exclusion chromatography as previously described (25). the time course of product formation was measured by HPLC as described for the single-dNTP assays. Analytical Size Exclusion Chromatography. Purified samples of SAMHD1c-RN μ The pair-wise dNTP mixture assays were performed with equal amount (2 mg/mL, 200 L) mixed with a final concentration of 4 mM dGTP or 4 mM dN1-dN2 of two dNTPs to obtain kapp , the apparent turnover rates for the dN1TP GTP were applied to a Superdex 200 10/300 GL column (GE Healthcare) mixture substrate with the dN2TP cofactor at Allo-site 2. The VdNi , the dNi (i = preequilibrated in 50 mM Tris·HCl, pH 8.0, 150 mM NaCl, 5 mM MgCl2, and

1,2) production rate in the dNTPs mixture, were measured. The identity of BIOCHEMISTRY 0.5 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP). The UV absor- the nucleotide (dN2TP) that occupies Allo-site 2 was determined from the bance at 280 nm was recorded for the elution of SAMHD1 oligomers. mixture results from our structure-based affinity assays. VdN2 and the apparent turnover rate kdN2-dN2, obtained in the single-dNTP activity assays, was used AUC. Sedimentation velocity experiments were performed with a Beckman XL-I app to calculate [SAMHD1dN1-dN2], the fraction of SAMHD1 that hydrolyzes analytical ultracentrifuge. Samples were prepared with protein concentration dN TP with dN TP at Allo-site 2 (Eq. 2–3). The kdN1-dN2 parameter was then of 1 mg/mL in the buffer containing 50 mM Tris·HCl, pH 8.0, 150 mM NaCl, 5 mM 1 2 app calculated as follows (Eq. 4): MgCl2, and 0.5 mM TCEP and equilibrated with a final concentration of 100 μM nucleotide. AUC was performed at 42,000 rpm and 20 °C with an An60-Ti rotor. h i mixture − VdN The experimental parameters including sample partial specific volume, buffer SAMHD1dN2 dN2 = 2 [2] dN2 −dN2 density, and viscosity were calculated with SEDNTERP (http://sednterp.unh. kapp edu/). Velocity data were analyzed using the program SEDFIT (36). h i h i − − SAMHD1dN1 dN2 = 1 − SAMHD1dN2 dN2 [3] Crystallization and Data Collection. SAMHD1c-RN in buffer (20 mM Tris·HCl, pH 7.8, 50 mM NaCl, 5 mM MgCl , and 2 mM DTT) was mixed with various combi- mixture 2 − V dN1 dN2 = dN1 nations of nucleotides (Table 1) and incubated at 4 °C for 30 min before crys- kapp − [4] ½SAMHD1dN1 dN2 tallization. Crystals were grown at 25 °C using the microbatch under-oil method by mixing 1 μL protein (5 mg/mL) with 1 μL crystallization buffer [100 mM SPG Similar analyses were performed for three- or four-dNTP (500 μM each) (Qiagen) buffer, pH 7.4, 25% polyethylene glycol 1500 (PEG 1500)]. Crystals were mixture assays. The production rates of dN were quantified and the dN TP cryoprotected by crystallization buffer supplemented with 25% (vol/vol) glycerol i 2 bound to Allo-site 2 was determined as above. The fractions of effective before frozen in liquid nitrogen. Diffraction data were collected at the Advanced dNi-dN2 SAMHD1 used by dNiTP substrates, [SAMHD1 ](i= 1,2,3,4), were Photon Source beamline 24-ID. The data statistics are summarized in Table S1. mixture then calculated using VdNi and the kapp values determined in the pair-wise dNTP mixture assays (Eq.5). If each k rate remained unchanged Structure Determination and Refinement. The structures were solved by mo- app in the various experiments and were correctly calculated, the sum of all lecular replacement using PHASER (37). The previously published SAMHD1 [SAMHD1dNi-dN2] should give 100% effective enzyme utilization, which can tetramer structure, with all bound nucleotides removed, was used as the therefore serve as a validation to the rates obtained in the assays. search model (PDB ID code 4BZB). The model was refined with iterative rounds of TLS (translation/libration/screw) and restrained refinement using h i Vmixture dNi −dN2 dNi Refmac5 (38) followed by rebuilding the model to the 2Fo-Fc and the Fo-Fc SAMHD1 = [5] dNi −dN2 maps using Coot (39). Refinement statistics are summarized in Table S1. kapp Coordinates and structure factors have been deposited in the Protein Data Bank, with accession codes listed in Table S1. ACKNOWLEDGMENTS. We thank X. Jia, J. Fribourgh, H. Nguyen, and E. Weber for assistance and discussion, K. S. Anderson and W. H. Konigsberg for comments, and M. Alonso, J. Deacon, R. Vithayathil, and S. Wojcik for technical SAMHD1 dNTPase Activity Assays. All SAMHD1 dNTPase activity assays were assistance. We also thank the staff at the Advanced Photon Source beamlines performed in a reaction buffer containing 50 mM Tris·HCl, pH 8.0, 150 mM NaCl, 24ID-C and E and the National Synchrotron Light Source beamlines X25. This 5mMMgCl2 and 0.5 mM TCEP. GTP, dNTP(s), and purified SAMHD1c were work was supported by National Institutes of Health Grant AI097064 (to Y.X.).

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