Allosteric regulation of the human and mouse deoxyribonucleotide triphosphohydrolase sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1)

Abstract

The deoxyribonucleotide triphosphohydrolase SAMHD1 restricts lentiviral infection by depleting the dNTPs required for viral DNA synthesis. In cultured human fibroblasts SAMHD1 is expressed maximally during quiescence preventing accumulation of dNTPs outside S phase. siRNA silencing of SAMHD1 increases dNTP pools, stops cycling human cells in G1, and blocks DNA replication. Surprisingly, knock-out of the mouse gene does not affect the well being of the animals. dNTPs are both substrates and allosteric effectors for SAMHD1. In the crystal structure each subunit of the homotetrameric protein contains one substrate-binding site and two nonidentical effector-binding sites, site 1 binding dGTP, site 2 dGTP or dATP. Here we compare allosteric properties of pure recombinant human and mouse SAMHD1. Both enzymes are activated 3-4-fold by allosteric effectors. We propose that in quiescent cells where SAMHD1 is maximally expressed GTP binds to site 1 with very high affinity, stabilizing site 2 of the tetrameric structure. Any canonical dNTP can bind to site 2 and activate SAMHD1, but in cells only dATP or dTTP are present at sufficient concentrations. The apparent Km for dATP at site 2 is ∼10 μm for mouse and 1 μm for human SAMHD1, for dTTP the corresponding values are 50 and 2 μm. Tetrameric SAMHD1 is activated for the hydrolysis of any dNTP only after binding of a dNTP to site 2. The lower Km constants for human SAMHD1 induce activation at lower cellular concentrations of dNTPs thereby limiting the size of dNTP pools more efficiently in quiescent human cells.

Keywords: Allosteric Regulation; DNA Enzyme; DNA Replication; Enzyme Kinetics; Enzyme Mechanism.

FIGURE 1.

SAMHD1 is a dNTP triphosphohydrolase. PAGE analysis and activity assay of purified recombinant mouse and human SAMHD1 are shown. A, [³H]dATP (100 μm, 30,000 cpm/nmol) and 100 ng of mouse SAMHD1 were incubated for 0 or 20 min under assay conditions. After protein precipitation the reaction mixture was chromatographed isocratically together with nonlabeled carrier deoxyadenosine and dATP as detailed under “Experimental Procedures.” The graph shows the radioactivity of the reaction products in 0.5-ml fractions collected at the two time points. B, the same experiment is shown but with 300 ng of human SAMHD1. The insets show the electrophoretic analyses of 4 μg of each enzyme and the positions of molecular size markers.

FIGURE 2.

Influence of substrate concentration on the dephosphorylation of ³H-labeled dNTPs by SAMHD1. Upper row, incubations with 50 ng of mouse SAMHD1; lower row, incubations with 150 ng of human SAMHD1. Each enzyme was incubated in separate experiments with ³H-labeled dGTP, dATP, dTTP, or dCTP (200–6000 cpm/nmol) at the varying concentrations shown on the abscissa and with 20 μm effectors (color-coded in the graphs). Other incubation conditions, the quantitation of the deoxynucleosides formed during the reaction, and the calculation of K_m constants are described under “Experimental Procedures.” Ordinates indicate the nmol of deoxynucleoside (NdR) produced in 1 min by 1 mg of protein.

FIGURE 3.

GTP and dGTP as effectors for the dephosphorylation of dATP and dTTP. Upper row, mouse SAMHD1; lower row, human SAMHD1. A, and B, dephosphorylation of 100 μm [³H]dATP by 50 ng of mouse SAMHD1 (A) or 150 ng of human SAMHD1 (B), at the GTP or dGTP concentrations shown on the abscissa. C, dephosphorylation of 100 μm [³H]dTTP by 150 ng of human SAMHD1. General conditions are as in Fig. 2. Ordinates show V − V_o where V is the rate of dephosphorylation in the presence of effector and V_o is the rate in its absence.

FIGURE 4.

dATP and dTTP as effectors for the dephosphorylation of dCTP or dTTP in the presence of GTP. Upper row, mouse SAMHD1; lower row, human SAMHD1. A and B, dephosphorylation of 30 μm [³H]dCTP at the dATP concentrations shown on the abscissa and in the presence of 20 or 500 μm GTP and 50 ng of mouse SAMHD1 (A) or 150 ng of human SAMHD1 (B), as described in Fig. 2. C, hydrolysis of 30 μm [³H]dTTP by 50 ng of mouse SAMHD1 with dATP as effector at the above two concentrations of GTP. D and E, dTTP as effector for the hydrolysis of 30 μm [³H]dCTP by 50 ng of mouse SAMHD1 (D) or by 150 ng of human enzyme (E) in the presence of 20 μm GTP.

FIGURE 5.

Cooperation between effectors during the dephosphorylation of dCTP or dTTP by SAMHD1. Upper row, mouse SAMHD1; lower row, human SAMHD1. A, [³H]dCTP as substrate. B, [³H]dTTP as substrate. We incubated each [³H]dNTP (30 μm = shaded bars, 100 μm = black bars) with 50 ng of mouse SAMHD1 and the indicated effectors (no eff, no effector added; G, GTP; dG, dGTP; dA, dATP; dC, dCTP; dT, dTTP) 10 μm each to determine the formation of labeled deoxycytidine ([³H]CdR) or thymidine (³H-TdR). General conditions were as described for Fig. 2. The ordinate shows the initial rate of each reaction (nmol of deoxyribonucleoside/min/mg of enzyme). C and D, the corresponding experiments with human SAMHD1, 150 ng/reaction, and all effectors at 3 μm.

FIGURE 6.

Competition between dGTP and GTP for allosteric site 1 during dephosphorylation of dATP by mouse SAMHD1. The experiment shows the changes in SAMHD1 activity by combining increasing concentrations of dGTP with a series of fixed concentrations of GTP as effectors for the dephosphorylation of 30 μm [³H]dATP by 50 ng of mouse SAMHD1. The concentration of GTP used in each curve is color-coded, the abscissa indicates the concentrations of dGTP. The data demonstrate the superiority of GTP as effector and the competition between dGTP and GTP at allosteric site 1. General conditions are as described for Fig. 2.

FIGURE 7.

Competition between dTTP and dATP for allosteric site 2 during dephosphorylation of dCTP by mouse SAMHD1. We determined the influence of 5 or 20 μm dATP on the dephosphorylation of 30 μm [³H]dCTP by 50 ng of mouse SAMHD1 in the presence of increasing concentrations of dTTP as effector. All reactions were carried out in the presence of 20 μm GTP under the general conditions given in Fig. 2. The data demonstrate the superiority of dATP over dTTP as effector and the competition of the two dNTPs for allosteric site 2.