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...

The sterile alpha motif and HD domain-containing protein 1 (SAMHD1), a dNTPase, prevents the infection of nondividing cells by retroviruses, including HIV, by depleting the cellular dNTP pool available for viral reverse transcription. SAMHD1 is a major regulator of cellular dNTP levels in mammalian cells. Mutations in SAMHD1 are associated with chronic lymphocytic leukemia (CLL) and the autoimmune condition Aicardi Goutières syndrome (AGS). The dNTPase activity of SAMHD1 can be regulated by dGTP, with which SAMHD1 assembles into catalytically active tetramers. Here we present extensive biochemical and structural data that reveal an exquisite activation mechanism of SAMHD1 via combined action of both GTP and dNTPs. We obtained 26 crystal structures of SAMHD1 in complex with different combinations of GTP and dNTP mixtures, which depict the full spectrum of GTP/dNTP binding at the eight allosteric and four catalytic sites of the SAMHD1 tetramer. Our data demonstrate how SAMHD1 is activated by binding of GTP or dGTP at allosteric site 1 and a dNTP of any type at allosteric site 2. Our enzymatic assays further reveal a robust regulatory mechanism of SAMHD1 activity, which bares resemblance to that of the ribonuclease reductase responsible for cellular dNTP production. These results establish a complete framework for a mechanistic understanding of the important functions of SAMHD1 in the regulation of cellular dNTP levels, as well as in HIV restriction and the pathogenesis of CLL and AGS.HIV restriction factor | dNTP metabolism | tetramerization | triphosphohydrolase | allosteric regulation S AMHD1 is a dNTPase that hydrolyzes dNTPs into deoxyribonucleosides (dNs) and triphosphates (1). It has recently been identified as a restriction factor that blocks infection by a broad range of retroviruses, including HIV-1, in noncycling myeloid-lineage cells and quiescent CD4 + T lymphocytes (2-7). The dNTPase activity of SAMHD1 depletes the cellular dNTP pool, inhibiting the reverse transcription of the viral RNA genome (8-10). SAMHD1 was also found to inhibit infection by certain DNA viruses including herpes simplex virus type 1 (HSV-1) and vaccinia virus in nondividing macrophages (11,12). Apart from viral restriction, SAMHD1 is ubiquitously expressed in both differentiated and undifferentiated cells of various human organs (2, 13), where it functions in the regulation of DNA damage signaling and proper activation of the innate immune response (14, 15). Mutations in SAMHD1, many of which result in deficiency in the dNTPase activity, are associated with chronic lymphocytic leukemia (CLL) (15, 16) and the autoimmune condition AicardiGoutieres syndrome (AGS) (17, 18).It has been recognized that SAMHD1 may play an important role in the regulation of cellular dNTP levels, which are critical to the fidelity of DNA synthesis and the stability of the genome (13). Abnormal size and/or the imbalance of the dNTP pool may activate the S-phase checkpoint for cell cycle arrest (19,20). In mammalian cells, the concentration of cellular dNT...

The recent explosive outbreak of Zika virus (ZIKV) infection has been reported in South and Central America and the Caribbean. Neonatal microcephaly associated with ZIKV infection has already caused a public health emergency of international concern. No specific vaccines or drugs are currently available to treat ZIKV infection. The ZIKV helicase, which plays a pivotal role in viral RNA replication, is an attractive target for therapy. We determined the crystal structures of ZIKV helicase-ATP-Mn2+ and ZIKV helicase-RNA. This is the first structure of any flavivirus helicase bound to ATP. Comparisons with related flavivirus helicases have shown that although the critical P-loop in the active site has variable conformations among different species, it adopts an identical mode to recognize ATP/Mn2+. The structure of ZIKV helicase-RNA has revealed that upon RNA binding, rotations of the motor domains can cause significant conformational changes. Strikingly, although ZIKV and dengue virus (DENV) apo-helicases share conserved residues for RNA binding, their different manners of motor domain rotations result in distinct individual modes for RNA recognition. It suggests that flavivirus helicases could have evolved a conserved engine to convert chemical energy from nucleoside triphosphate to mechanical energy for RNA unwinding, but different motor domain rotations result in variable RNA recognition modes to adapt to individual viral replication.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-016-0293-2) contains supplementary material, which is available to authorized users.

Significance COVID-19 is a deadly rampaging infectious disease with over 480 million cases worldwide. Unfortunately, effective therapies remain very limited. Novel antiviral agents are urgently needed to combat this global healthcare crisis. Here, we elucidate the structural basis for replicase polyprotein cleavage and substrate specificity of SARS-CoV-2 main protease (M pro ). Through analyzing a series of high-resolution structures of SARS-CoV-2 M pro throughout the proteolytic process, we demonstrate the molecular mechanism of M pro in proteolytic processing that confers substrate specificity. Substrate selectivity is revealed using structures of the H41A mutant in complex with six individual native cleavage substrates. Our study underscores the mechanistic function of M pro in the viral life cycle, which provides structural insights to develop effective inhibitors against this essential target of SARS-CoV-2.

Background: Phosphorylation of SAMHD1 Thr-592 inhibits its anti-HIV activity. Results: Phosphomimetic mutation T592E of SAMHD1 perturbs SAMHD1 crystal structure, destabilizes SAMHD1 tetramer, and reduces its dNTP triphosphatase (dNTPase) activity. Conclusion: T592E decreases the dNTPase activity of SAMHD1 via destabilizing the catalytically active tetramer. Significance: Structure-induced impairment of SAMHD1 dNTPase activity by T592E suggests a mechanism of the phosphorylation-regulated SAMHD1 antiviral activity.