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...

Background: SAMHD1, a dGTP-activated dNTPase, inhibits retrovirus infection at the reverse transcription step in monocytes and quiescent T lymphocytes.Results: dGTP-induced SAMHD1 tetramerization correlates with its functional activation.Conclusion: SAMHD1 tetramers are the biologically active form of this dNTPase.Significance: Learning how SAMHD1 function is regulated is important for understanding innate and anti-viral immunity.

Background: Human SAMHD1 protein restricts HIV/SIV infection of myeloid cells and is targeted for proteasomal degradation by HIV-2 Vpx protein.Results: Vpx binds the divergent C terminus of human SAMHD1 and loads it onto DCAF1 substrate receptor of CRL4 E3 ubiquitin ligase.Conclusion: Vpx programs SAMHD1 degradation by loading it onto CRL4DCAF1.Significance: Learning how viruses overcome innate anti-viral mechanisms is critical for the conception of new antiviral therapeutics.

The cellular process of intrinsic apoptosis relies on the peroxidation of mitochondrial lipids as a critical molecular signal. Lipid peroxidation is connected to increases in mitochondrial reactive oxygen species, but there is also a required role for mitochondrial cytochrome c (cyt-c). In apoptotic mitochondria, cyt-c gains a new function as a lipid peroxidase that catalyzes the reactive oxygen species-mediated chemical modification of the mitochondrial lipid cardiolipin (CL). This peroxidase activity is caused by a conformational change in the protein, resulting from interactions between cyt-c and CL. The nature of the conformational change and how it causes this gain-of-function remain uncertain. Via a combination of functional, structural, and biophysical experiments we investigate the structure and peroxidase activity of cyt-c in its membrane-bound state. We reconstituted cyt-c with CL-containing lipid vesicles, and determined the increase in peroxidase activity resulting from membrane binding. We combined these assays of CL-induced proapoptotic activity with structural and dynamic studies of the membrane-bound protein via solid-state NMR and optical spectroscopy. Multidimensional magic angle spinning (MAS) solid-state NMR of uniformly (13)C,(15)N-labeled protein was used to detect site-specific conformational changes in oxidized and reduced horse heart cyt-c bound to CL-containing lipid bilayers. MAS NMR and Fourier transform infrared measurements show that the peripherally membrane-bound cyt-c experiences significant dynamics, but also retains most or all of its secondary structure. Moreover, in two-dimensional and three-dimensional MAS NMR spectra the CL-bound cyt-c displays a spectral resolution, and thus structural homogeneity, that is inconsistent with extensive membrane-induced unfolding. Cyt-c is found to interact primarily with the membrane interface, without significantly disrupting the lipid bilayer. Thus, membrane binding results in cyt-c gaining the increased peroxidase activity that represents its pivotal proapoptotic function, but we do not observe evidence for large-scale unfolding or penetration into the membrane core.

The HIV-1 accessory protein Vpr is required for efficient viral infection of macrophages and promotion of viral replication in T cells. Vpr’s biological activities are closely linked to the interaction with human DCAF1, a cellular substrate receptor of the Cullin4–RING E3 ubiquitin ligase (CRL4) of the host ubiquitin–proteasome-mediated protein degradation pathway. The molecular details of how Vpr usurps the protein degradation pathway have not been delineated. Here we present the crystal structure of the DDB1–DCAF1–HIV-1–Vpr–uracil-DNA glycosylase (UNG2) complex. The structure reveals how Vpr engages with DCAF1, creating a binding interface for UNG2 recruitment, in a manner distinct from the recruitment of SAMHD1 by Vpx protein for degradation by Vpx proteins. Vpr and Vpx use similar N-terminal and helical regions to bind the substrate receptor, whereas different regions target the specific cellular substrates. Furthermore, Vpr uses molecular mimicry of DNA by a variable loop for specific recruitment of the UNG2 substrate.

Background: Human sterile ␣ motif and histidine-aspartate domain-containing protein 1 (SAMHD1) is a deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase that is phosphorylated by cyclinA2-dependent kinases. Results: SAMHD1 mutants defective for cyclinA2 binding disrupt S phase progression, and this is alleviated by Thr-592 phosphomimetic mutation. Conclusion: CyclinA2-dependent kinases regulate SAMHD1 activity. Significance: SAMHD1 dNTP phosphohydrolase activity is regulated during the cell cycle.

Background: SAMHD1 is a deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase. Results: SAMHD1 forms a catalytically active tetramer upon binding of two nucleoside triphosphates with different specificities at two adjacent allosteric sites. Conclusion: The primary allosteric site selectively binds guanine-containing nucleotides, whereas the secondary site accommodates any dNTP. Significance: The tetramerization and catalytic activity of SAMHD1 is differentially regulated by different nucleoside triphosphates.

813transitions is that allowing for orbital relaxation lowers the predicted excitation energies, generally by 0.3-0.5 eV. Thus transition-state excitation energies would probably be very close in absolute value to those observed experimentally. This additional evidence that the calculations are consistent with experiment further increases our confidence in our previously presented2 conclusions about bonding in Rh2C12(C0)4.HF LCAO Calculations of Rh2Cl2(CO),. Rh,C12(C0)4 has recently been treated theoretically by a nonempirical Hartree-Fock LCAO method, using a pseudopotential to represent the core orbitals.' Insofar as the discussion in this paper parallels our own work, there appears to be good agreement between the two calculations. In the folded conformation, a Mulliken population analysis of the LCAO orbitals yields charges of Rh-O.l*, C l P 3 , C+0.3B, and O-'.26. We can obtain atomic charges from the Xa-SW orbitals by normalizing the total number of valence electrons found within the atomic spheres to the total number possessed by the molecule;1s this gives Rh*.09, C14.15, C+0.46, and O-0.34. It is encouraging to see such close agreement between two very different methods of estimating charge.The H F LCAO ordering for the mainly Rh 4d orbitals is identical with ours; there are minor differences in the ordering of the mainly C13p orbitals. The other authors do not discuss the possibility of a weak Rh-Rh bonding interaction in the mainly C13p orbitals, which we found to be the reason for the folding of the molecule. They do report a result which we did not, namely, that the folded form is calculated to be 4.1 kcal/mol more stable than the planar form. Given the close agreement between the two calculations in other respects, wewould not be surprised if an analysis of the H F LCAO mainly C1 orbitals also revealed an enhanced Rh-Rh interaction upon folding. Experimental Section Rh2CIz(C0)4 was prepared by the method of WilkinsonI6 by reaction of RhC13-xH20 with C O at ca. 90 OC. The sample was purified by vacuum sublimation at 80 OC mmHg) and the IR spectrum checked prior to recording the He I and He I1 PE spectra. Rh2-C12(PF3)4 was prepared from Rh2Cl2(CZH4)2' by treatment with PF31* and purified by sublimation at 20 OC mmHg), and the I9F N M R and IR spectra were checked prior to recording the PE spectrum.He I photoelectron spectra were recorded on a PS16 Perkin-Elmer spectrometer. The Rh2C12(PF3)4 was vaporized into the instrument via an external inlet held at room temperature. RhzClz(C0)4 was vaporized internally at 60 * 1 OC.The spectra were recorded several times over a 6-h period and in all cases the reproducibility was excellent. No traces of free PF3 or C O were observed under our conditions. (16) J.The electron density distribution of iron pyrite, FeS2, has been determined from high-resolution single-crystal X-ray diffraction measurements. Data collected with Ag Ka radiation to a resolution of (sin @)/A = 1.46 A-' were refined by conventional least-squares techniques to R = 1.8% and R, = 1.5%. Pyrite c...