GTP activator and dNTP substrates of HIV-1 restriction factor SAMHD1 generate a long-lived activated state

Abstract

The HIV-1 restriction factor sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) is a tetrameric protein that catalyzes the hydrolysis of all dNTPs to the deoxynucleoside and tripolyphosphate, which effectively depletes the dNTP substrates of HIV reverse transcriptase. Here, we establish that SAMHD1 is activated by GTP binding to guanine-specific activator sites (A1) as well as coactivation by substrate dNTP binding to a distinct set of nonspecific activator sites (A2). Combined activation by GTP and dNTPs results in a long-lived tetrameric form of SAMHD1 that persists for hours, even after activating nucleotides are withdrawn from the solution. These results reveal an ordered model for assembly of SAMHD1 tetramer from its inactive monomer and dimer forms, where GTP binding to the A1 sites generates dimer and dNTP binding to the A2 and catalytic sites generates active tetramer. Thus, cellular regulation of active SAMHD1 is not determined by GTP alone but instead, the levels of all dNTPs and the generation of a persistent tetramer that is not in equilibrium with free activators. The significance of the long-lived activated state is that SAMHD1 can remain active long after dNTP pools have been reduced to a level that would lead to inactivation. This property would be important in resting CD4 + T cells, where dNTP pools are reduced to nanomolar levels to restrict infection by HIV-1. dNTP induced oligomerization | enzyme catalysis | innate immunity T he steady-state composition and concentration of deoxynucleotide triphosphate pools in mammalian cells are highly regulated because of the mutagenic consequences of dNTP imbalances in dividing cells (1, 2) as well as the important antiviral effects of dNTP pool depletion in quiescent cells (3,4). In all cell types, the ultimate pool balance is determined by dNTP-dependent regulatory pathways that affect the activities of enzymes involved in both synthesis and degradation of dNTPs (5-7). The most important highly up-regulated synthetic enzyme during S phase of dividing cells is the R1/R2 isoform of ribonucleotide triphosphate reductase, which ensures that dNTP precursors are plentiful for DNA synthesis (8). However, in quiescent cells of the immune system (resting CD4 + T cells, macrophages, and dendritic cells), where dNTP pools are ∼10-fold lower than dividing cells, the ultimate pool levels are likely determined by a balance between the activities of the R1/p53R2 isoform of ribonucleotide triphosphate reductase and the degradative dNTP triphosphohydrolase sterile α-motif/histidineaspartate domain-containing protein 1 (SAMHD1) (9). The highly dynamic nature of dNTP pools demands finely tuned mechanisms for feedback regulation of these enzymes by dNTPs as well as coarse regulatory mechanisms (posttranslational modifications, transcriptional regulation, and proteasomal targeting) that serve to turn these activities on and off at appropriate stages of the cell cycle and in specific cell types (10, 11). dNTP triphosphohydrolase enzymes, such as SAMHD1,...

Structural basis of cellular dNTP regulation by SAMHD1

“…The reaction profiles were fitted to a sigmoidal kinetic function, which resulted in various degrees of sigmoidal shaped curves expected from cooperativity effect due to allosteric binding. The dGTP reaction showed the lowest cooperativity, which is consistent with that observed previously (27). In contrast, the dCTP reaction profile showed the most pronounced sigmoidal behavior of activation.…”

“…Our study provides a structural basis and complementary analysis to the reported study on dGTP/dUTP catalysis by SAMHD1 (27). During the preparation of this manuscript, Hansen et al (27) reported the combined activation of SAMHD1 tetramer by GTP and dUTP.…”

“…This approach is possible because our previous biochemical data indicate that SAMHD1 tetramers are stable and may perform multiple catalytic cycles without having to go through the dimer-tetramer transition (28). This approach is also supported by the recent report that the activated state of SAMHD1 is long-lived even when dNTP levels are subsequently reduced to very low levels (27). We preincubated SAMHD1c with GTP (500 μM) and a specific dNTP (dN2, 500 μM) to form the active tetramer.…”

“…Although the cellular dGTP concentration may provide a convenient 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.…”

“…Structural and biochemical studies revealed that SAMHD1 was activated by allosteric dGTPinduced tetramerization, which shapes the substrate-binding pocket for dNTP binding and catalysis (25). Interestingly, recent enzymatic studies suggested that GTP is the major activator of SAMHD1 (26) and dNTPs substrates may also be involved in activating the enzyme (27), indicating a regulation mechanism more complex than previously thought.…”