Mechanism of allosteric activation of SAMHD1 by dGTP

Abstract

SAMHD1, a dNTP triphosphohydrolase (dNTPase), has a key role in human innate immunity. It inhibits infection of blood cells by retroviruses, including HIV, and prevents the development of the autoinflammatory Aicardi-Goutières syndrome (AGS). The inactive apo-SAMHD1 interconverts between monomers and dimers, and in the presence of dGTP the protein assembles into catalytically active tetramers. Here, we present the crystal structure of the human tetrameric SAMHD1-dGTP complex. The structure reveals an elegant allosteric mechanism of activation through dGTP-induced tetramerization of two inactive dimers. Binding of dGTP to four allosteric sites promotes tetramerization and induces a conformational change in the substrate-binding pocket to yield the catalytically active enzyme. Structure-based biochemical and cell-based biological assays confirmed the proposed mechanism. The SAMHD1 tetramer structure provides the basis for a mechanistic understanding of its function in HIV restriction and the pathogenesis of AGS.

Figure 1. Crystal structure of the SAMHD1c2 tetramer in complex with dGTPs

(a) Surface representations of the tetramer in three orthogonal views. Subunits A, B, C and D are colored in light blue, pale cyan, red and wheat, respectively. (b) Left, the dimer-2 interface between subunits A (ribbon) and C (transparent surface). Regions that are disordered in the SAMHD1c1 structure are highlighted in magenta. Right, the previously observed dimer-1 interface between subunits A (ribbon) and D (transparent surface). Bound dGTP molecules are depicted in stick representations with the catalytic site dGTPs in cyan and the allosteric site dGTPs in orange. A schematic cartoon of the tetramer is provided in the middle box using the same color scheme as in the rest of the figure. (c) Putty representation of SAMHD1c2 subunit in two orthogonal views. The structure of SAMHD1c2 has been aligned to SAMHD1c1 using SHP. The color spectrum and the coil thickness represent the deviation of the aligned Cα atoms (RMSD) in the two structures, which varies from 1 Å (blue) to 7.4 Å (orange). The disordered regions (residues 506–515, 531–547, 584–599, with each end labeled) in SAMHD1c1 are colored red and displayed with large thickness. Residues 113–119 which are not included in SAMHD1c1 are marked in black. (d) Dimer-2 (tetramer) interface with the most variable regions.

Figure 2. dGTP–Mg²⁺–dGTP binding at the allosteric site induces tetramerization of SAMHD1

(a) Left, the SAMHD1c2 tetramer in surface representation, using the same color scheme as in Figure 1. Right, the allosteric dGTP binding site between subunits A, C, and D with the D subunit shown in semi-transparent ribbon representation to clearly illustrate the buried dGTP–Mg²⁺–dGTP (yellow and orange sticks). The yellow sphere represents a magnesium ion. Residues 113–119 of subunit D are shown as a black ribbon. (b) Interactions between the two dGTP molecules and their surrounding residues. Residues are colored differently for each subunit, using the same color scheme as in Figure 1. Hydrogen bonds are shown as dashed lines. (c) The dimer-2 interface between subunits A (light blue ribbon) and C (grey surface). Residues that are not observed in the previous SAMHD1c1 dimer structure are highlighted in magenta. The three layers of interactions at the AC interface are boxed. The black oval and line indicate a 2-fold symmetry axis. (b–d) Details of the interactions in the three different layers, first (b), second (c), and third (d), respectively. Several key residues are shown in stick representation. The black oval in the center represents a two-fold axis perpendicular to the given view.

Figure 3. Mutagenesis of SAMHD1 residues involved in dGTP binding and tetramerization

(a) Quantification of SAMHD1 tetramer by analytical size exclusion chromatography, expressed as percentage of the total protein. The experiments were performed in duplicates and the average values are shown. (b) in vitro dNTPase assay of wild type (WT) and mutant SAMHD1 proteins. The nucleoside product (dA) was quantified by HPLC. Each experiment was performed in triplicate. Error bars show the standard deviations (s. d.). (c) HIV-1 restriction and (d) intracellular dNTPase activities of WT SAMHD1, and the allosteric dGTP-binding site and the tetramer interface mutants. Luciferase activity in U937 cell extracts was quantified and is shown in arbitrary units. dATP pools in U937 cells are presented in pmol/10⁶ cells. WT and mutant SAMHD1 were detected by immunoblotting for the N-terminal epitope tag and α-tubulin was used as a loading control. U937 cells transduced with an empty MSCV (puro) vector (M) were used as a negative control. Luciferase reporter activity or dNTP levels in cells expressing WT or mutant SAMHD1 proteins are plotted as a function of protein expression levels, normalized to that of the highest expression level of WT SAMHD1. The values of luciferase activity and dATP amounts shown are averages of three replicates. Error bars, s. d.. Similar results were obtained in two independent experiments. Original images of blots used in this study can be found in Supplementary Figure 6.

Figure 4. Comparison of SAMHD1c2 and SAMHD1c1 structures at the catalytic site

(a) Superposition of subunits of tetrameric SAMHD1c2 (light blue) and dimeric SAMHD1c1 (tan). Subunits A and C are shown in ribbon and surface representation, respectively. The dGTP substrate is depicted in green and the metal ion as a green sphere. Missing regions in SAMHD1c1 are highlighted in magenta in the SAMHD1c2 structure. (b) Conformational changes in the substrate binding pocket flanked by the α-helix at the dimer-2 interface. Key residues are shown in stick representation with hydrogen bonds shown as dotted lines. (c) Comparison of the substrate binding pockets (surface representation) in the two structures. The catalytic residues are shown on the left and key contact residues are shown on the right. The dGTP (grey) in the dimer is modeled based on the position in the tetramer. (d) dATPase assay for WT SAMHD1 and substrate binding site mutants. Each experiment was performed in triplicate. Error bars, s. d.

Figure 5. Schematic illustration of the mechanism of SAMHD1 activation by allosteric dGTP-induced tetramerization

Subunits of SAMHD1 are labeled A, B, C and D and the catalytically inactive monomer and dimers are shown with empty substrate binding pockets. Binding of four dGTP pairs at the allosteric sites promotes tetramerization and induces large conformational changes at the tetramer interface (AC and BD). This, in turn, causes conformational changes around the catalytic sites enabling dNTP substrate binding in a catalytically productive conformation.