title: Effects of T592 phosphomimetic mutations on tetramer stability and dNTPase activity of SAMHD1 can not explain the retroviral restriction defect.
abstract: SAMHD1, a dNTP triphosphohydrolase, contributes to interferon signaling and restriction of retroviral replication. SAMHD1-mediated retroviral restriction is thought to result from the depletion of cellular dNTP pools, but it remains controversial whether the dNTPase activity of SAMHD1 is sufficient for restriction. The restriction ability of SAMHD1 is regulated in cells by phosphorylation on T592. Phosphomimetic mutations of T592 are not restriction competent, but appear intact in their ability to deplete cellular dNTPs. Here we use analytical ultracentrifugation, fluorescence polarization and NMR-based enzymatic assays to investigate the impact of phosphomimetic mutations on SAMHD1 tetramerization and dNTPase activity in vitro. We find that phosphomimetic mutations affect kinetics of tetramer assembly and disassembly, but their effects on tetramerization equilibrium and dNTPase activity are insignificant. In contrast, the Y146S/Y154S dimerization-defective mutant displays a severe dNTPase defect in vitro, but is indistinguishable from WT in its ability to deplete cellular dNTP pools and to restrict HIV replication. Our data suggest that the effect of T592 phosphorylation on SAMHD1 tetramerization is not likely to explain the retroviral restriction defect, and we hypothesize that enzymatic activity of SAMHD1 is subject to additional cellular regulatory mechanisms that have not yet been recapitulated in vitro.
authors: Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229, USA.
authors: Akash
authors: A
authors: Bhattacharya
authors: Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229, USA.
authors: Zhonghua
authors: Z
authors: Wang
authors: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors: Tommy
authors: T
authors: White
authors: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors: Cindy
authors: C
authors: Buffone
authors: Center for Drug Discovery, Department of Pediatrics, Emory School of Medicine, Atlanta, GA 30322, USA.
authors: Laura A
authors: LA
authors: Nguyen
authors: Center for Drug Discovery, Department of Pediatrics, Emory School of Medicine, Atlanta, GA 30322, USA.
authors: Caitlin N
authors: CN
authors: Shepard
authors: Center for Drug Discovery, Department of Pediatrics, Emory School of Medicine, Atlanta, GA 30322, USA.|School of Pharmacy, Kyunghee University, Seoul, South Korea.
authors: Baek
authors: B
authors: Kim
authors: Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229, USA.
authors: Borries
authors: B
authors: Demeler
authors: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors: Felipe
authors: F
authors: Diaz-Griffero
authors: Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229, USA.
authors: Dmitri N
authors: DN
authors: Ivanov
mesh_terms: D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D002460:Cell Line; D006678:HIV / Q000502:physiology*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism*; D010766:Phosphorylation; D055503:Protein Multimerization; D000076106:SAM Domain and HD Domain-Containing Protein 1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000235:genetics*
chemical_list: D009711:Nucleotides; D014443:Tyrosine; D000076106:SAM Domain and HD Domain-Containing Protein 1; C484280:SAMHD1 protein, human