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GTP Activator and Dntp Substrates of HIV-1 Restriction Factor SAMHD1

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GTP Activator and Dntp Substrates of HIV-1 Restriction Factor SAMHD1 GTP activator and dNTP substrates of HIV-1 restriction PNAS PLUS factor SAMHD1 generate a long-lived activated state Erik C. Hansen, Kyle J. Seamon, Shannen L. Cravens, and James T. Stivers1 Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205-2185 Edited by Myron F. Goodman, University of Southern California, Los Angeles, CA, and accepted by the Editorial Board March 26, 2014 (received for review January 27, 2014) The HIV-1 restriction factor sterile α-motif/histidine-aspartate phatases (14). Presumably, degradation to the level of nucleoside domain-containing protein 1 (SAMHD1) is a tetrameric protein that rather than deoxynucleoside diphosphate (dNDP) or deoxy- catalyzes the hydrolysis of all dNTPs to the deoxynucleoside and nucleoside monophosphate (dNMP) is to make the process tripolyphosphate, which effectively depletes the dNTP substrates energetically or kinetically costly to reverse, with the additional of HIV reverse transcriptase. Here, we establish that SAMHD1 is possibility that the neutral nucleoside will be irreversibly trans- activated by GTP binding to guanine-specific activator sites (A1) as ported out of the cell (9). The unusual triphosphohydrolase activity well as coactivation by substrate dNTP binding to a distinct set of of SAMHD1 in quiescent immune cells has received significant nonspecific activator sites (A2). Combined activation by GTP and attention, because HIV-1 and HSV-1 are severely restricted in their dNTPs results in a long-lived tetrameric form of SAMHD1 that ability to infect quiescent cells that have severely depressed dNTP persists for hours, even after activating nucleotides are withdrawn pools, which has been directly linked to SAMHD1 enzymatic ac- from the solution. These results reveal an ordered model for as- tivity (15, 16). Mutations in the SAMHD1 gene have also been sembly of SAMHD1 tetramer from its inactive monomer and dimer linked to Aicardi–Goutières syndrome, a rare genetic autoim- forms, where GTP binding to the A1 sites generates dimer and mune encephalopathy with a chronic inflammatory pathology that dNTP binding to the A2 and catalytic sites generates active tetra- resembles congenital viral infections (17). As would be expected, + mer. Thus, cellular regulation of active SAMHD1 is not deter- resting CD4 T cells from Aicardi–Goutières syndrome patients mined by GTP alone but instead, the levels of all dNTPs and the are permissive to HIV-1 infection (18). generation of a persistent tetramer that is not in equilibrium A key question with SAMHD1 is how its activity is tuned in with free activators. The significance of the long-lived activated response to dNTP pool levels. Early reports established that the state is that SAMHD1 can remain active long after dNTP pools dNTPase activity of SAMHD1 required the presence of dGTP as have been reduced to a level that would lead to inactivation. an activator when dATP, dTTP, or dCTP was used as a substrate This property would be important in resting CD4+ Tcells,where (15, 17). Subsequently, it was reported that the dGTP activator/ dNTP pools are reduced to nanomolar levels to restrict infection substrate can be replaced by GTP, which serves as an activator, by HIV-1. but not a substrate (19). Two recent structures of SAMHD1 in complex with dNTPs show a tetrameric quaternary structure with BIOCHEMISTRY dNTP induced oligomerization | enzyme catalysis | innate immunity dGTP molecules bound to the (A1A2)4 activator sites and one dNTP bound to each of four catalytic sites (20). Comparison with he steady-state composition and concentration of deoxy- an earlier dimeric structure of the free enzyme suggests that Tnucleotide triphosphate pools in mammalian cells are highly activator and/or substrate binding drive the enzyme into the regulated because of the mutagenic consequences of dNTP tetrameric form (15, 21). imbalances in dividing cells (1, 2) as well as the important The importance of this enzyme in innate immunity (15), auto- antiviral effects of dNTP pool depletion in quiescent cells (3, 4). immunity (17), and control of the transition from G1 to S phase In all cell types, the ultimate pool balance is determined by in the cell cycle (5) makes an understanding of its regulatory and dNTP-dependent regulatory pathways that affect the activities of enzymes involved in both synthesis and degradation of dNTPs Significance (5–7). The most important highly up-regulated synthetic enzyme during S phase of dividing cells is the R1/R2 isoform of ribo- The degradative dNTP triphosphohydrolase activity of the nucleotide triphosphate reductase, which ensures that dNTP sterile α-motif/histidine-aspartate domain-containing protein 1 precursors are plentiful for DNA synthesis (8). However, in (SAMHD1) enzyme helps maintain optimal dNTP balances for + quiescent cells of the immune system (resting CD4 T cells, DNA replication and also serves as an HIV-1 restriction factor in + macrophages, and dendritic cells), where dNTP pools are ∼10-fold resting CD4 target cells of HIV by depleting dNTP substrates of lower than dividing cells, the ultimate pool levels are likely de- reverse transcriptase. This study shows that full activation of termined by a balance between the activities of the R1/p53R2 SAMHD1 involves ordered binding of GTP and substrate dNTPs isoform of ribonucleotide triphosphate reductase and the deg- to activator and substrate sites on the enzyme, leading to or- radative dNTP triphosphohydrolase sterile α-motif/histidine- dered assembly of the tetramer active form. After the enzyme aspartate domain-containing protein 1 (SAMHD1) (9). The highly is activated, it no longer communicates with free activator dynamic nature of dNTP pools demands finely tuned mechanisms nucleotides, which contributes to efficient depletion of dNTP for feedback regulation of these enzymes by dNTPs as well as pools in resting T cells. coarse regulatory mechanisms (posttranslational modifications, Author contributions: E.C.H., K.J.S., S.L.C., and J.T.S. designed research; E.C.H., K.J.S., and transcriptional regulation, and proteasomal targeting) that serve S.L.C. performed research; E.C.H., K.J.S., S.L.C., and J.T.S. analyzed data; and E.C.H., K.J.S., to turn these activities on and off at appropriate stages of the cell S.L.C., and J.T.S. wrote the paper. cycle and in specific cell types (10, 11). The authors declare no conflict of interest. dNTP triphosphohydrolase enzymes, such as SAMHD1, are This article is a PNAS Direct Submission. M.F.G. is a guest editor invited by the conserved from bacteria to humans and carry out the unusual Editorial Board. conversion of dNTPs to the nucleoside (dN) and tripolyphosphate 1To whom correspondence should be addressed. E-mail: [email protected]. (12, 13). The tripolyphosphate is likely degraded to 3Pi through This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the action of plentiful cellular pyrophosphatases or tripolyphos- 1073/pnas.1401706111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1401706111 PNAS | Published online April 21, 2014 | E1843–E1851 Downloaded by guest on September 30, 2021 KdUTP = : ± : catalytic mechanism of interest. In addition, such mechanistic giving a limiting m 1 5 0 1 mM at saturating GTP activa- KGTP = ± μ V dUTP=½ = kdUTP = work can also provide useful insights to guide the design of mech- tor with act 163 30 Mand max monomers cat ± : −1 V dUTP anism-based activators and inhibitors of SAMHD1 that could have 5 0 2s . Normalization of max to monomer concentration is research or therapeutic uses. In this study, we have characterized the justified, because plots of velocity against SAMHD1 monomer coordinated effects of activator and substrate binding on oligomer- concentration were linear in the range of 0.2–10 μMwhenthe ization and activation of the enzyme and discovered a long-lived GTP activator concentration was ≥50 μM. (Fig. S2). app;dUTP activated state of SAMHD1 that is not in equilibrium with free ac- A secondary plot of Km against 1/[GTP] activator tivator nucleotides. We propose that GTP combined with any dNTP concentration clearly shows that the catalytic site has un- serve as the preferred activators for SAMHD1 in the cell and that detectable affinity for dUTP in the absence of GTP (Fig. 1D) app;dUTP the long-lived activated state is key for efficient dNTP depletion. and that GTP acts to lower the Km without changing app;dUTP Vmax (Fig. S3A). Therefore, GTP is a Km-type essential acti- Results vator, and high dUTP substrate concentrations always pull the Activation of dUTP Hydrolysis by GTP. The complex dGTP/GTP- equilibrium to the active enzyme-activator-substrate (EAS) com- dependent activation mechanism for dNTP hydrolysis and the plex, even at low activator concentrations. The cellular implication possibility that SAMHD1 might exist in multiple active oligomeric of this mechanism is that, as long as the activator concentration states present a challenging system for designing kinetic experi- exceeds that of the enzyme, dNTP binding can, in principle, pull ments that reveal useful insights into function and mechanism. the system all of the way to the activated EAS state. Because activation by dGTP is complicated by the fact that it is To confirm the ordered essential activation mechanism, we also a substrate, we began by investigating activation by GTP, also performed a kinetic analysis, where dUTP hydrolysis was which does not compete for substrate binding to the catalytic site. measured at fixed dUTP concentrations (0.2–5 mM) and the The activation of dUTP hydrolysis by GTP was studied using GTP activator concentration was varied in the range of 0.01– 3 A B E =Kapp;GTP H-labeled dUTP (Fig. 1 and ). dUTP was selected for these 5 mM (Fig. 1 ). As expected, the secondary replot of 1 act studies, because it is present at high concentrations in resting against [dUTP] was linear, with the y intercept providing the true KGTP = ± μ F immune cells that express SAMHD1 and it reacts similarly as all act 133 10 M (Fig.
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