Phospho-dependent Regulation of SAMHD1 Oligomerisation Couples Catalysis and Restriction

Abstract

SAMHD1 restricts HIV-1 infection of myeloid-lineage and resting CD4+ T-cells. Most likely this occurs through deoxynucleoside triphosphate triphosphohydrolase activity that reduces cellular dNTP to a level where reverse transcriptase cannot function, although alternative mechanisms have been proposed recently. Here, we present combined structural and virological data demonstrating that in addition to allosteric activation and triphosphohydrolase activity, restriction correlates with the capacity of SAMHD1 to form “long-lived” enzymatically competent tetramers. Tetramer disruption invariably abolishes restriction but has varied effects on in vitro triphosphohydrolase activity. SAMHD1 phosphorylation also ablates restriction and tetramer formation but without affecting triphosphohydrolase steady-state kinetics. However phospho-SAMHD1 is unable to catalyse dNTP turnover under conditions of nucleotide depletion. Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity where dephosphorylation switches housekeeping SAMHD1 found in cycling cells to a high-activity stable tetrameric form that depletes and maintains low levels of dNTPs in differentiated cells.

A Highly Active Isoform of Lentivirus Restriction Factor SAMHD1 in Mouse

“…ISF1/ISF2, because it has only a single carboxyl-terminal domain from ISF2, is dGTP-dependent. This model agrees with that proposed by Arnold et al (31) who used ddGTP, a substrate that is hydrolyzed by SAMHD1 but does not bind the allosteric site, to show that the carboxylterminal domain phosphorylation does not affect SAMHD1 catalytic activity in presence of dGTP yet destabilizes the tetramer in the absence of the activator. The model predicts that ISF2, because of its high stability, can deplete the dNTP pool effectively in non-cycling cells where the rate of dNTP synthesis is low (Fig.…”

SAMHD1: Recurring roles in cell cycle, viral restriction, cancer, and innate immunity

“…The sequential activation and assembly of the SAMHD1 tetramer occur at dNTP concentrations multiple orders of magnitude below the K M . Thus, while reported K M values for SAMHD1 vary considerably from 50–350 μM depending on the study, standard Michaelis-Menten kinetic models apply under all the experimental conditions measured (55, 57, 73, 78, 81, 82). Given the steady-state conditions of an activated SAMHD1 tetramer, the turnover rate for each substrate appears to depend solely on its particular affinity for the active site and its intracellular concentration.…”

“…Some evidence suggests phosphorylation negatively modulates SAMHD1 tetramerization and dNTPase activity (81, 99, 127), and this diminished dNTPase capacity is responsible for increased intracellular dNTP pools (93, 120, 121, 128). Other studies find limited or no effect of phosphorylation of SAMHD1 on catalytic activity(81, 108, 116, 117, 129), oligomerization equilibrium and allosteric activation(77, 129), or nucleic acid binding (83). Complicating the model further, phosphorylation may affect SAMHD1 substrate specificity (78).…”

“…P-T592 is also less likely to form the activated tetramer at low concentration of activating nucleotides (78, 81). Structural data support these findings by identifying protein conformational shifts induced by a phosphomimetic mutant (T592E) that destabilizes the tetramer interface (127).…”

“…Closer inspection of SAMDH1 crystal structures reveal that C522 comprises one member of a cysteine triad - C522, C341, and C350 - that resides adjacent to the regulatory nucleotide binding site RS2. In several available crystal structures (pdbid: 5AO4, 4RXO, 4MZ7, 3U1N)(54, 70, 74, 81), a disulfide bond exists between C341 and C350 and results in a conformational shift that may disrupt activating nucleotide binding and tetramer stability. While the experiments in the study were performed using recombinant human SAMHD1, it is interesting to note that the residues comprising the cysteine triad (C522, C341, and C350) are highly conserved among vertebrate SAMHD1 homologues.…”