Mechanism of allosteric activation of SAMHD1 by dGTP

Abstract

SAMHD1, a deoxyribonucleoside triphosphate triphosphohydrolase (dNTPase), plays 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 via 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.The sterile alpha motif and HD-domain containing protein 1 (SAMHD1) dNTPase plays dual roles in human innate immunity. It restricts HIV-1 infection in immune cells of myeloid Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms Accession codesThe coordinates and structure factors have been deposited in PDB, with accession code 4BZC for the wild type and 4BZB for the RN mutant. HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript lineage and in quiescent CD4-positive T lymphocytes [1][2][3][4][5] . In these non-dividing cells, SAMHD1 reduces cellular dNTP levels to concentrations below the threshold required for reverse transcription of the viral RNA genome into DNA 6-8 . Furthermore, mutations in SAMHD1 are associated with an autoimmune condition, termed Aicardi Goutières Syndrome (AGS) 9,10 , whose clinical manifestations resemble congenital viral infection 11,12 . AGS-associated SAMHD1 mutations appear to disrupt the dNTPase activity of SAMHD1. Thus, SAMHD1's ability to negatively regulate cellular dNTP levels is essential for its roles in innate immunity 13,14 .The dNTPase activity of SAMHD1 resides in its histidine-aspartate (HD) domain, with the N-terminal sterile alpha motif (SAM) domain involved in other activities [13][14][15][16][17] . A recent crystal structure of a dimeric SAMHD1 catalytic core fragment (SAMHD1c1, residues 120-626) suggested an allosteric, dGTP-stimulated mechanism for the promotion of the dNTPase activity of dimeric SAMHD1 14 . However, the SAMHD1c1 structure did not contain substrate or the dGTP cofactor, thus providing limited insight into the mechanism of SAMHD1 activation. Recent biochemical and functional studies revealed that SAMHD1 interconverts between an inactive monomeric or dimeric form and a dGTP-induced tetr...

GTP activator and dNTP substrates of HIV-1 restriction factor SAMHD1 generate a long-lived activated state

“…We found that racemic dGTPαS was a very slow substrate in the absence and presence of transactivation by GTP (t 1/2 = 24 h) (Fig. S5), which is consistent with the crystal structure showing that dGTPαS can bind to the A1 and A2 activator sites as well as the catalytic sites (20). Supporting the suggestion that dGTPαS can self-activate, it also served as a weak transactivator for dUTP hydrolysis at low concentrations (K dGTPαS act = 8 ± 2 μM and k dUTP cat = 0:9 ± 0:1 s −1 ) and an inhibitor at higher concentrations ðK dGTPαS i = 67 ± 19 μMÞ (Fig.…”

“…Unlike GTP, dGTP can activate its own hydrolysis and is known to serve as a transactivator for hydrolysis of other substrate dNTPs at low concentrations (17). However, it should also act as an inhibitor of dNTP hydrolysis at higher concentrations (20,22). To explore these properties of dGTP, we determined the concentration dependence of the initial rates of dGTP hydrolysis at an SAMHD1 concentration of 0.2 μM and variable concentrations of dGTP in the range of 5 μM to 5 mM ( Fig.…”

“…The crystal structure of SAMHD1 bound to 2′-deoxyguanosine-5′-[α-thio] triphosphate lithium salt (dGTPαS) shows that the R stereoisomer of this analog occupies all activator sites and catalytic sites of the tetramer, suggesting that it should serve as a stable dGTP analog for mechanistic studies (20). We found that racemic dGTPαS was a very slow substrate in the absence and presence of transactivation by GTP (t 1/2 = 24 h) (Fig.…”

“…Subsequently, it was reported that the dGTP activator/ substrate can be replaced by GTP, which serves as an activator, but not a substrate (19). Two recent structures of SAMHD1 in complex with dNTPs show a tetrameric quaternary structure with dGTP molecules bound to the (A1A2) 4 activator sites and one dNTP bound to each of four catalytic sites (20). Comparison with an earlier dimeric structure of the free enzyme suggests that activator and/or substrate binding drive the enzyme into the tetrameric form (15,21).…”

“…2C). These properties of dGTPαS, which may underestimate its potency by a factor of two because of the use of a racemic mixture, make it useful for comparing the discrete effect of a nonsubstrate activator (GTP) with that of activators that are known to bind to all activator and substrate sites of SAMHD1 (dGTP and dGTPαS) (20,22).…”