Probing the catalytic mechanism and inhibition of SAMHD1 using the differential properties of Rp- and Sp-dNTPαS diastereomers

DOI: 10.1021/acs.biochem.0c00944

Authors: Elizabeth R. Morris (The Francis Crick Institute) , Simone Kunzelmann (The Francis Crick Institute) , Sarah J. Caswell (The Francis Crick Institute (Midland Road)) , Andrew G. Purkiss (The Francis Crick Institute) , Geoff Kelly (The Francis Crick Institute) , Ian A. Taylor (The Francis Crick Institute)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochemistry

State: Published (Approved)
Published: May 2021
Diamond Proposal Number(s): 13775

Abstract: 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-Goutières 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 Rp and Sp diastereomers of dNTPαS nucleotides. Our biochemical and enzymological data show that the α-phosphorothioate substitution in Sp-dNTPαS but not Rp-dNTPαS 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 Sp-dNTPαS diastereomers competitively inhibit dNTP hydrolysis, while Rp-dNTPαS 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, Rp-dGTPαS, in which an Fe–Mg-bridging water species is poised for nucleophilic attack on the Pα. We conclude that it is the incompatibility of Mg2+, a hard Lewis acid, and the α-phosphorothioate thiol, a soft Lewis base, that prevents the Sp-dNTPαS nucleotides coordinating in a catalytically productive conformation. On the basis of these data, we present a model for SAMHD1 stereospecific hydrolysis of Rp-dNTPαS nucleotides and for a mode of competitive inhibition by Sp-dNTPαS nucleotides that competes with formation of the enzyme–substrate complex.

Journal Keywords: Monomers; Hydrolysis; Ions; Inhibition; Molecular structure

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I04-Macromolecular Crystallography

Added On: 21/05/2021 20:07

Documents:
acs.biochem.0c00944.pdf

Discipline Tags:

Biochemistry Catalysis Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)