Probing the Catalytic Mechanism and Inhibition of SAMHD1 Using the Differential Properties of Rp- and Sp-dNTPαS Diastereomers

SAMHD1 is a fundamental regulator of cellular dNTPs that catalyzes their hydrolysis into 2'-deoxynucleoside and triphosphate, restricting the replication of viruses, including HIV-1, in CD4(+) myeloid lineage and resting T-cells. SAMHD1 mutations are associated with the autoimmune disease Aicardi-Goutieres syndrome (AGS) and certain cancers. More recently, SAMHD1 has been linked to anticancer drug resistance and the suppression of the interferon response to cytosolic nucleic acids after DNA damage. Here, we probe dNTP hydrolysis and inhibition of SAMHD1 using the R-p and S-p diastereomers of dNTP alpha S nucleotides. Our biochemical and enzymological data show that the alpha-phosphorothioate substitution in S-p-dNTP alpha S but not R-p-dNTP alpha S diastereomers prevents Mg2+ ion coordination at both the allosteric and catalytic sites, rendering SAMHD1 unable to form stable, catalytically active homotetramers or hydrolyze substrate dNTPs at the catalytic site. Furthermore, we find that S-p-dNTP alpha S diastereomers competitively inhibit dNTP hydrolysis, while R-p-dNTP alpha S nucleotides stabilize tetramerization and are hydrolyzed with similar kinetic parameters to cognate dNTPs. For the first time, we present a cocrystal structure of SAMHD1 with a substrate, R-p-dGTP alpha S, in which an Fe-Mg-bridging water species is poised for nucleophilic attack on the P-alpha. We conclude that it is the incompatibility of Mg2+, a hard Lewis acid, and the alpha-phosphorothioate thiol, a soft Lewis base, that prevents the S-p-dNTP alpha S nucleotides coordinating in a catalytically productive conformation. On the basis of these data, we present a model for SAMHD1 stereospecific hydrolysis of R-p-dNTP alpha S nucleotides and for a mode of competitive inhibition by S-p-dNTP alpha S nucleotides that competes with formation of the enzyme-substrate complex.

Automated summary

SAMHD1 is a crucial factor in regulating cellular dNTPs and can suppress the replication of viruses like HIV-1 in specific cells, with mutations leading to autoimmune diseases and cancers. Experimental data shows that S-p-dNTP alpha S nucleotides competitively inhibit dNTP hydrolysis, while R-p-dNTP alpha S nucleotides stabilize tetramerization with similar catalytic parameters.