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Mechanism of allosteric activation of SAMHD1 by dGTP

Nature Structural & Molecular Biology volume 20, pages 1304–1309 (2013)Cite this article

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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.

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Figure 1: Crystal structure of the SAMHD1c2 tetramer in complex with dGTPs.
Figure 2: dGTP–Mg2+–dGTP binding at the allosteric site induces tetramerization of SAMHD1.
Figure 3: Mutagenesis of SAMHD1 residues involved in dGTP binding and tetramerization.
Figure 4: Comparison of SAMHD1c2 and SAMHD1c1 structures at the catalytic site.
Figure 5: Schematic illustration of the mechanism of SAMHD1 activation by allosteric dGTP-induced tetramerization.

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Acknowledgements

We thank X. Jia, E. Weber, J. Fribourgh, H. Nguyen and B. Summers for assistance and discussion and Y. Zuo, Y. Li and C. Wang for technical assistance. We also thank the staff at the Advanced Photon Source beamline 24-ID and the National Synchrotron Light Source beamline X25. We thank N. Landau (New York University School of Medicine), T. Hope (Northwestern University) and D. McDonald (Case Western Reserve University) for supplying reagents. This work was supported in part by funds from the University of Pittsburgh School of Medicine (J.A.) and by US National Institutes of Health grants AI097064 (Y.X.), AI100673 (J.S.) and P50GM82251 (A.M.G. and J.A.).

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Author notes

  1. Haitao Yang

    Present address: Present address: School of Life Sciences, Tianjin University, Tianjin, China.,

  2. Xiaoyun Ji, Ying Wu and Junpeng Yan: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA

    Xiaoyun Ji, Haitao Yang & Yong Xiong

  2. Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

    Ying Wu, Jennifer Mehrens, Maria DeLucia, Angela M Gronenborn & Jinwoo Ahn

  3. Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

    Ying Wu, Jennifer Mehrens, Maria DeLucia, Angela M Gronenborn, Jacek Skowronski & Jinwoo Ahn

  4. Department of Molecular Biology and Microbiology, Case Western Reserve School of Medicine, Cleveland, Ohio, USA

    Junpeng Yan, Caili Hao & Jacek Skowronski

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Contributions

X.J. performed protein crystallization, structure determination and structure analysis; X.J. and H.Y. collected the diffraction data; Y.W., J.M., M.D. and J.A. performed the biochemical mutation experiments and data analysis in vitro; J.Y. and C.H. performed the cell-based experiments; J.A. provided experimental materials; X.J., Y.W., J.Y., H.Y., A.M.G., J.S., J.A. and Y.X. analyzed the data; X.J., Y.W., J.S., J.Y., J.A. and Y.X. designed the experiments; and X.J., H.Y., A.M.G., J.S., J.A. and Y.X. wrote the manuscript.

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Correspondence to Jinwoo Ahn or Yong Xiong.

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Integrated supplementary information

Supplementary Figure 1 Structure comparison of SAMHD1c2 and SAMHD1c1.

(a) Structure superposition of SAMHD1c1 and SAMHD1c2. The structure of SAMHD1c2 has been aligned to SAMHD1c1 using SHP1. The SAMHD1c1 missing region is highlighted in red in the SAMHD1c2 structure. (b) Putty representation of the dimer-1 interface with little altered conformation. The color spectrum and the coil thickness represent the deviation of the aligned Cα atoms in the two structures, which varies from rmsd 1 Å (blue) to rmsd 7.4 Å (orange). The disordered regions in SAMHD1c1 are colored red and displayed with large thickness. Residues 113–119 which are not included in SAMHD1c1 are marked in black.

Supplementary Figure 2 2D representation of dGTP–Mg2+–dGTP interactions at the allosteric site.

dGTPs and SAMHD1 interaction residues are shown as lines and hydrogen bonds are shown as black dashed lines with distances marked. Residues were colored according to subunit: A – light blue, C – light red, D – wheat. The magnesium ion and a water molecule are shown as spheres. The interactions were analyzed and plotted by using Ligplot2.

Supplementary Figure 3 dGTPs at the allosteric site.

(a) Modeling of GTPs at the allosteric site. The dGTP-binding pocket at the allosteric site is shown in surface representation and the SAMHD1 subunits are colored the same as in Figure 1. GTPs are placed at the positions of dGTP-1 and dGTP-2 sites as black sticks. Residues V117 and F157 were drawn as sticks to show the steric hindrance to the 2'-hydroxyl group of the ribose. Binding of GTPs at the allosteric site would require a conformational change involving these residues. (b) The geometry of dGTP–Mg2+–dGTP at the allosteric site. The magnesium ion binds to five oxygen atoms of the β or γ phosphate groups from both of the two dGTP molecules and one ordered water in an octahedral geometry. dGTPs are shown as sticks. The Mg2+ ion (yellow) and water (red) are shown as spheres. The 2Fo–Fc electron density maps (σ=1.5) are shown as meshes. The Mg-O bonds were indicated by dotted lines.

Supplementary Figure 4 The phosphorylation site of SAMHD1.

Dimer-2 (tetramer) interface is shown between SAMHD1c2 subunit A (light blue, ribbon and surface) and C (red, surface). The disordered regions in SAMHD1c1 subunit A are shown as magenta ribbon. The phosphorylation target motif 592TPQK595 is shown in two orthogonal views as ribbon and surface representation in yellow.

Supplementary Figure 5 The catalytic site of SAMHD1.

The protein is shown in a cut-open surface representation (light blue). The dGTP substrate in the substrate-binding pocket is shown as sticks. Key interaction residues are shown as sticks (light blue) and marked. The metal ion is shown as a yellow sphere coordinated by side chains from four residues (H167, H206, D207 and D311) and the α-phosphate oxygen of the substrate. The catalytic triad (D218, H210 and H233) is displayed in the left panel. Metal-coordinating and hydrogen bonds are indicated by dashed lines. The 2′-hydroxyl group of a ribose in a NTP molecule would clash (marked as a magenta cross in the right panel) with Y374 and L150 in the tight binding pocket.

Supplementary Figure 6 Uncropped western blot images shown in Figure 3.

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Ji, X., Wu, Y., Yan, J. et al. Mechanism of allosteric activation of SAMHD1 by dGTP. Nat Struct Mol Biol 20, 1304–1309 (2013). https://doi.org/10.1038/nsmb.2692

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