SAMHD1 shapes deoxynucleotide triphosphate homeostasis by interconnecting the depletion and biosynthesis of different dNTPs

SAMHD1 is a dNTPase that impedes replication of HIV-1 in myeloid cells and resting T lymphocytes. Here we elucidate the substrate activation mechanism of SAMHD1, which involves dNTP binding at allosteric sites and transient tetramerization. Our findings reveal that tetramerization alone is insufficient to promote dNTP hydrolysis; instead, the activation mechanism requires an inactive tetrameric intermediate with partially occupied allosteric sites. The equilibrium between inactive and active tetrameric states regulates dNTPase activity, driven by the binding and dissociation of additional allosteric dNTP ligands to the preassembled tetramer. Furthermore, catalytic efficiency, but not substrate specificity, is modulated by the identity of the dNTPs occupying the allosteric sites. We show how this allosteric regulation shapes deoxynucleotide homeostasis by balancing dNTP production and SAMHD1-catalyzed depletion. Notably, SAMHD1 exhibits a distinct functionality, which we term facilitated dNTP depletion, whereby increased biosynthesis of certain dNTPs enhances the depletion of others. The regulatory relationship between the biosynthesis and depletion of different dNTPs sheds light on the emerging role of SAMHD1 in the biology of dNTP homeostasis with implications for HIV/AIDS, innate antiviral immunity, T cell disorders, telomere maintenance and therapeutic efficacy of nucleoside analogs.