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 re- actions 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.

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