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. 2016 Oct 7;291(41):21407-21413.
doi: 10.1074/jbc.C116.751446. Epub 2016 Aug 26.

Allosteric Activation of SAMHD1 Protein by Deoxynucleotide Triphosphate (dNTP)-dependent Tetramerization Requires dNTP Concentrations That Are Similar to dNTP Concentrations Observed in Cycling T Cells

Zhonghua Wang  1 , Akash Bhattacharya  1 , Jessica Villacorta  1 , Felipe Diaz-Griffero  2 , Dmitri N Ivanov  3
Affiliations

Allosteric Activation of SAMHD1 Protein by Deoxynucleotide Triphosphate (dNTP)-dependent Tetramerization Requires dNTP Concentrations That Are Similar to dNTP Concentrations Observed in Cycling T Cells

Zhonghua Wang et al. J Biol Chem. .

Abstract

SAMHD1 is a dNTP hydrolase, whose activity is required for maintaining low dNTP concentrations in non-cycling T cells, dendritic cells, and macrophages. SAMHD1-dependent dNTP depletion is thought to impair retroviral replication in these cells, but the relationship between the dNTPase activity and retroviral restriction is not fully understood. In this study, we investigate allosteric activation of SAMHD1 by deoxynucleotide-dependent tetramerization and measure how the lifetime of the enzymatically active tetramer is affected by different dNTP ligands bound in the allosteric site. The EC50dNTP values for SAMHD1 activation by dNTPs are in the 2-20 μm range, and the half-life of the assembled tetramer after deoxynucleotide depletion varies from minutes to hours depending on what dNTP is bound in the A2 allosteric site. Comparison of the wild-type SAMHD1 and the T592D mutant reveals that the phosphomimetic mutation affects the rates of tetramer dissociation, but has no effect on the equilibrium of allosteric activation by deoxynucleotides. Collectively, our data suggest that deoxynucleotide-dependent tetramerization contributes to regulation of deoxynucleotide levels in cycling cells, whereas in non-cycling cells restrictive to retroviral replication, SAMHD1 activation is likely to be achieved through a distinct mechanism.

Keywords: autoimmune disease; human immunodeficiency virus (HIV); innate immunity; nucleoside/nucleotide metabolism; retrovirus.

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Figures

FIGURE 1.
FIGURE 1.
FP studies of SAMHD1 tetramer lifetime. A, SAMHD1 tetramerization requires nucleotide binding at the allosteric sites A1 and A2. When GTP-F is incorporated into SAMHD1 tetramers formed in the presence of unlabeled GTP and dNTPs, FP readings increase. Deoxynucleotides are then rapidly depleted by the enzyme, and the gradual decay of FP values reflects the rate of tetramer dissociation. Dissociation rates were obtained by fitting baseline-corrected FP readings to a simple exponential decay equation (black lines) as explained under “Experimental Procedures.” B, the tetramer dissociation rate of the dATP-bound tetramer is significantly faster for the T592D phosphomimetic mutant than for the WT SAMHD1 construct. C, in contrast, in the dCTP-bound SAMHD1 tetramer, the dissociation rate is slower for the T592D mutant. D, in comparison with the dATP- and dCTP-bound tetramers, the dTTP-mediated SAMHD1 tetramerization displays lower signal amplitude and faster apparent dissociation rates. Error bars indicate means ± S.E.
FIGURE 2.
FIGURE 2.
Lifetime measurements of the enzymatically active SAMHD1 tetramers using a non-equilibrium dNTPase assay. A, the enzymatically active SAMHD1 tetramer is formed after incubation with GTP and dNTPs. A non-equilibrium state of the enzyme can subsequently be created using a desalting spin column. Tetramer dissociation is observed as a gradual decrease of the dTTPase hydrolysis rate as a function of the incubation time as described under “Results”. B–E, decay rates (koff) of the SAMHD1 enzymatic activity vary between 10−4 and 10−2 s−1 depending on the dNTP bound at the A2 site. The effect of the T592D mutation is also dNTP-dependent. The solid and dashed lines show the best fits of the data to the simple exponential decay equation. Error bars indicate means ± S.E.
FIGURE 3.
FIGURE 3.
Measurement of the EC50dNTP constants for allosteric activation of SAMHD1 by deoxynucleotides. A, modification of the non-equilibrium dNTPase assay that allows measurement of the EC50dNTP values. dNTP concentrations in the initial incubation are varied, and the fraction of enzymatically active SAMHD1 is determined by measuring the dNTP hydrolysis rate. B–E, EC50dNTP values for all nucleotides are in the low micromolar range, and the effect of the T592D mutation is 2-fold or less. The solid and dashed lines show the best fits of the data to the simple EC50 equation. Error bars indicate means ± S.E.
FIGURE 4.
FIGURE 4.
Implications for SAMHD1 function. A, a predominantly kinetic effect of the T592D mutation is most likely explained by a change in the activation energy for the rate-limiting step in the SAMHD1 tetramerization process. The mutation has only a minor effect on the tetramerization equilibrium. B, EC50dNTP values for SAMHD1 activation by deoxynucleotides match dNTP concentrations in cycling immune cells. The nucleotide-dependent tetramerization of SAMHD1 may thus contribute to dNTP regulation in cycling cells. SAMHD1 activation in non-cycling cells is likely to be mediated by a distinct mechanism.
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