The missing link: allostery and catalysis in the anti-viral protein SAMHD1

Abstract

Vertebrate protein SAMHD1 (sterile-α-motif and HD domain containing protein 1) regulates the cellular dNTP (2'-deoxynucleoside-5'-triphosphate) pool by catalysing the hydrolysis of dNTP into 2'-deoxynucleoside and triphosphate products. As an important regulator of cell proliferation and a key player in dNTP homeostasis, mutations to SAMHD1 are implicated in hypermutated cancers, and germline mutations are associated with Chronic Lymphocytic Leukaemia and the inflammatory disorder Aicardi-Goutières Syndrome. By limiting the supply of dNTPs for viral DNA synthesis, SAMHD1 also restricts the replication of several retroviruses, such as HIV-1, and some DNA viruses in dendritic and myeloid lineage cells and resting T-cells. SAMHD1 activity is regulated throughout the cell cycle, both at the level of protein expression and post-translationally, through phosphorylation. In addition, allosteric regulation further fine-tunes the catalytic activity of SAMHD1, with a nucleotide-activated homotetramer as the catalytically active form of the protein. In cells, GTP and dATP are the likely physiological activators of two adjacent allosteric sites, AL1 (GTP) and AL2 (dATP), that bridge monomer-monomer interfaces to stabilise the protein homotetramer. This review summarises the extensive X-ray crystallographic, biophysical and molecular dynamics experiments that have elucidated important features of allosteric regulation in SAMHD1. We present a comprehensive mechanism detailing the structural and protein dynamics components of the allosteric coupling between nucleotide-induced tetramerization and the catalysis of dNTP hydrolysis by SAMHD1.

Keywords: HD domain; HIV; SAMHD1; allosteric regulation; hydrolase; oligomerization.

Figure 1.. SAMHD1 structure and catalytic activity.

(A) The dNTP triphosphohydrolysis reaction catalysed by SAMHD1, dGTP is shown as the example substrate. (B) Domain organisation of human SAMHD1, showing the nuclear localisation signal in blue, the SAM domain in dark orange, the HD domain in light orange, the HD motif residues in maroon and phosphorylated residue Thr592 in teal. (C) Left: NMR structure of the human SAMHD1 SAM domain (PDB code: 2E8O residues 34–113). Right: X-ray crystal structure of WT human SAMHD1 HD domain (PDB code: 4BZC, monomer A, residues 115–599) [85]. The structures of the two domains are connected by a short, dotted and grey line. The HD motif-co-ordinated manganese ion is shown as a purple sphere, and the HD motif residues are shown as maroon sticks. (D) A close-up view of the HD motif-co-ordinated manganese ion in the catalytic site. PyMOL was used to prepare the structural figures.

Figure 2.. Nucleotide-dependent tetramerization of SAMHD1 HD domain.

(A) Ordered pathway for tetramer assembly, proposed by Hansen et al. [97], and elucidated structurally using X-ray crystallography [,,–88,94,95,101]. The four protein monomers of SAMHD1 are shown in light orange, grey, maroon and light green. From left to right: The apo SAMHD1 HD domain is in a monomer–dimer equilibrium with GTP (dark blue), which stabilises dimerisation by coordinating AL1 (PDB code: 4RXO) [95]. dATP (light blue) co-ordinates AL2, and a magnesium ion (green sphere) bridges adjacent AL1–AL2 allosteric sites to stabilise the HD domain tetramer (PDB code: 4RXP) [95]. dCTP in the catalytic site is shown in pink. (B) GTP–dATP coordination at adjacent allosteric sites, AL1–AL2, of an HD domain tetramer (PDB code: 4TO0) [87]. GTP in AL1 and dATP in AL2 are shown in stick representation, with C_α atoms shown in yellow. Hydrogen-bonding interactions are represented by dashed lines.

Figure 3.. Thr592 phosphorylation modulates SAMHD1 tetramer stability.

(A) X-ray crystal structure of SAMHD1 HD domain tetramer (PDB code: 4BZC) [85]. Monomer A is shown in cartoon representation in light orange. Monomers B–D are shown in surface representation in grey. Residues 559–599 are in pink and residues 522–537 are in blue. (B) C-terminal, the α-helical region between residues 559–599, comprising phosphorylated residue Thr592.

Figure 4.. SAMHD1 catalytic site accommodates dNTPs and dNTP analogues.

(A–F) Nucleotide coordination in the catalytic site of the human SAMHD1 HD domain. (A) WT SAMHD1 with dGTPαS (PDB: 4BZC) [85]. (B) H206R/D207N SAMHD1 with dATP (PDB: 4QG1) [88]. (C) H206R/D207N SAMHD1 with TTP (PDB: 4TNZ) [87]. (D) WT SAMHD1 with dCTP (PDB: 4RXR) [95]. (E) WT SAMHD1 with ddGTP (PDB: 5AO1) [77]. (F) H206R/D207N SAMHD1 with gemcitabine-TP (Gem-TP) (PDB: 6DW5) [101]. Nucleotides are shown in stick representation with C_α atoms in pink. Manganese (purple), magnesium (green) and iron (brown) metal ions are represented as spheres.

Figure 5.. ‘Open’ and ‘closed’ conformations of HD domain.

(A) X-ray crystal structure of the HD domain in ‘open’ conformation co-ordinated to GTP (dark blue) in AL1 (PDB: 4RXO) [95]. The tetramer comprises two dimers, with one formed from the orange and grey monomers, and the second from the maroon and light green monomers. (B) X-ray crystal structure of HD domain in ‘closed’ tetrameric conformation, with dGTP co-ordinated in AL1, AL2 and the catalytic site (PDB: 4BZB) [85]. dGTP is shown in dark blue (AL1), pale blue (AL2) or pink (catalytic site) and magnesium ions are shown as green spheres. (C) Superposition of an HD domain monomer in ‘open’ (grey) and ‘closed’ (orange) conformations. Residues 326–375 and 454–599 differ in their conformation between the ‘open’ and ‘closed’ states and are represented by darker shades of grey and orange, respectively. The dGTP- and magnesium-coordination observed in the catalytic site of the ‘closed’ state is displayed, while the manganese- and phosphate-coordination observed in the ‘open’ state is not shown. (D) Interactions formed by the α₁₃ helix at the dimer–dimer interface (residues Asn358, Asp361, His364 and Arg372), in the catalytic site (Arg366, Tyr374 and Gln375) and in AL2 (Arg352, Lys354 and Asn358). One monomer is shown in pale orange, and its neighbouring monomer at the dimer–dimer interface is shown in maroon. dGTP in AL1 (dark blue), AL2 (pale blue) and the catalytic site (pink) is shown in stick representation. Magnesium ions are represented by green spheres. Hydrogen bonds are shown as dashed lines.