Uncovering allostery and regulation in SAMHD1 through molecular dynamics simulations

Abstract

The human sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a retroviral restriction factor in myeloid cells and non-cycling CD4+ T cells, a feature imputed to its phosphohydrolase activity-the enzyme depletes the cellular dNTP levels inhibiting reverse transcription. The functionally active form of SAMHD1 is an allosterically triggered tetramer which utilizes GTP-Mg -dNTP cross bridges to link and stabilize adjacent monomers. However, very little is known about how it assembles into a tetramer and how long the tetramer stays intact. In this computational study, we provide a molecular dynamics based analysis of the structural stability and allosteric site dynamics in SAMHD1. We have investigated the allosteric links which assemble and hold the tetramer together. We have also extended this analysis to a regulatory mutant of SAMHD1. Experimental studies have indicated that phosphorylation of T592 downregulates HIV-1 restriction. A similar result is also achieved by a phosphomimetic mutation T592E. While a mechanistic understanding of the process is still elusive, the loss of structural integrity of the enzyme is conjectured to be the cause of the impaired dNTPase activity of the T592E mutant. MD simulations show that the T592E mutation causes slightly elevated local motions which remain confined to the short helix (residues 591-595), which contains the phosphorylation site and do not cause long-range destabilization of the SAMHD1 tetramer within the timeframe of the simulations. Thus, the regulatory mechanism of SAMHD1 is a more subtle mechanism than has been previously suspected. Proteins 2017; 85:1266-1275. © 2017 Wiley Periodicals, Inc.

Molecular dynamics investigation of a redox switch in the anti‐HIV protein SAMHD1

“…Two sets of independent trajectories of length 200 and 300 ns each were generated from MD calculations at 295 K for each of the eight monomeric systems. In addition, the trajectories of the wt tetrameric system (with C341 and C350 disulfide linked) that were generated in our previous study were also used for analysis. Here, we selected a particular monomer (chain B) from the tetrameric system for comparison with the other monomeric systems that were investigated in this study.…”

“…in vivo tetramer‐lifetime biochemical investigations demonstrated that SAMHD1 assembly and activation requires a K m of ~10 μM, which is the dNTP concentration in cycling cells . Our own computational studies shed light on the influence of GTP activators and dNTP substrate on tetramer stability and allosteric pathways within the tetramer . However, this model is applicable only in cycling cells, whereas it is in terminally differentiated cells such as macrophages that SAMHD1 restricts HIV‐1.…”

SAMHD1 Functions and Human Diseases

“…An active tetramer, then, is stabilized by four allosteric sites. Each allosteric site contacts three monomers in the structure and contains a dGTP/GTP-Mg 2+ -dNTP bridge [22]. Additional metal ions are chelated by the His167-His206-Asp207-Asp311 quartet in order to correctly orient and polarize the dNTP substrate in the catalytic sites of each monomer.…”

“…The identification of cell cycle-regulated phosphorylated SAMHD1 resulted in the quest to characterize this post-translational modification and its effect on the tetramerization and activation of SAMHD1. While many groups found that dNTP activators dissociate from the A2 allosteric sites of pSAMHD1, leading to tetramer destabilization and subsequent loss of triphosphohydrolase activity [22,25,35,36], there are conflicting reports stating that the dNTPase activity of pSAMHD1 and phosphomimetic mutants T592E and T592D are very similar to wildtype SAMHD1 [37][38][39][40]. Despite the discrepancies surrounding the dNTPase activity of pSAMHD1, phosphorylation of SAMHD1 has also been shown to play a role in HIV-1 restriction-a function that we will further discuss in later sections.…”

SAMHD1 … and Viral Ways around It

“…Oligomerization of SAMHD1 is required for its catalytic dNTPase activity and is induced upon cofactor binding at two allosteric sites within the protein [7][8][9][10][11][12]. At allosteric site 1, dGTP or GTP binding leads to SAMHD1 dimerization, while the subsequent binding of any dNTP to allosteric site 2 induces tetramer formation [1,13,14]. SAMHD1 converts dNTPs into deoxynucleosides (dN) and inorganic triphosphates (PPPi) and thereby counteracts the de novo dNTP synthesis, primarily conducted by ribonucleotide reductase (RNR) and cellular deoxynucleoside kinases, which are mainly active during the S phase of the cell cycle [15][16][17][18].…”