Ian A. Taylor

HIV-1 restriction factor SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase

SAMHD1, an analogue of the murine interferon (IFN)-γ-induced gene Mg11 (ref. 1), has recently been identified as a human immunodeficiency virus-1 (HIV-1) restriction factor that blocks early-stage virus replication in dendritic and other myeloid cells and is the target of the lentiviral protein Vpx, which can relieve HIV-1 restriction. SAMHD1 is also associated with Aicardi–Goutières syndrome (AGS), an inflammatory encephalopathy characterized by chronic cerebrospinal fluid lymphocytosis and elevated levels of the antiviral cytokine IFN-α. The pathology associated with AGS resembles congenital viral infection, such as transplacentally acquired HIV. Here we show that human SAMHD1 is a potent dGTP-stimulated triphosphohydrolase that converts deoxynucleoside triphosphates to the constituent deoxynucleoside and inorganic triphosphate. The crystal structure of the catalytic core of SAMHD1 reveals that the protein is dimeric and indicates a molecular basis for dGTP stimulation of catalytic activity against dNTPs. We propose that SAMHD1, which is highly expressed in dendritic cells, restricts HIV-1 replication by hydrolysing the majority of cellular dNTPs, thus inhibiting reverse transcription and viral complementary DNA (cDNA) synthesis.

The Molecular Basis of FHA Domain:Phosphopeptide Binding Specificity and Implications for Phospho-Dependent Signaling Mechanisms

Forkhead-associated (FHA) domains are a class of ubiquitous signaling modules that appear to function through interactions with phosphorylated target molecules. We have used oriented peptide library screening to determine the optimal phosphopeptide binding motifs recognized by several FHA domains, including those within a number of DNA damage checkpoint kinases, and determined the X-ray structure of Rad53p-FHA1, in complex with a phospho-threonine peptide, at 1.6 A resolution. The structure reveals a striking similarity to the MH2 domains of Smad tumor suppressor proteins and reveals a mode of peptide binding that differs from SH2, 14-3-3, or PTB domain complexes. These results have important implications for DNA damage signaling and CHK2-dependent tumor suppression, and they indicate that FHA domains play important and unsuspected roles in S/T kinase signaling mechanisms in prokaryotes and eukaryotes.

Structure and catalytic mechanism of the human histone methyltransferase SET7/9

Acetylation, phosphorylation and methylation of the amino-terminal tails of histones are thought to be involved in the regulation of chromatin structure and function. With just one exception, the enzymes identified in the methylation of specific lysine residues on histones (histone methyltransferases) belong to the SET family. The high-resolution crystal structure of a ternary complex of human SET7/9 with a histone peptide and cofactor reveals that the peptide substrate and cofactor bind on opposite surfaces of the enzyme. The target lysine accesses the active site of the enzyme and the S-adenosyl-l-methionine (AdoMet) cofactor by inserting its side chain into a narrow channel that runs through the enzyme, connecting the two surfaces. Here we show from the structure and from solution studies that SET7/9, unlike most other SET proteins, is exclusively a mono-methylase. The structure indicates the molecular basis of the specificity of the enzyme for the histone target, and allows us to propose a model for the methylation reaction that accounts for the role of many of the residues that are invariant across the SET family.

High-resolution structure of a retroviral capsid hexameric amino-terminal domain

Retroviruses are the aetiological agents of a range of human diseases including AIDS and T-cell leukaemias. They follow complex life cycles, which are still only partly understood at the molecular level. Maturation of newly formed retroviral particles is an essential step in production of infectious virions, and requires proteolytic cleavage of Gag polyproteins in the immature particle to form the matrix, capsid and nucleocapsid proteins present in the mature virion. Capsid proteins associate to form a dense viral core that may be spherical, cylindrical or conical depending on the genus of the virus. Nonetheless, these assemblies all appear to be composed of a lattice formed from hexagonal rings, each containing six capsid monomers. Here, we describe the X-ray structure of an individual hexagonal assembly from N-tropic murine leukaemia virus (N-MLV). The interface between capsid monomers is generally polar, consistent with weak interactions within the hexamer. Similar architectures are probably crucial for the regulation of capsid assembly and disassembly in all retroviruses. Together, these observations provide new insights into retroviral uncoating and how cellular restriction factors may interfere with viral replication.

Key features of the interaction between Pcf11 CID and RNA polymerase II CTD

The C-terminal domain (CTD) of the large subunit of RNA polymerase II is a platform for mRNA processing factors and links gene transcription to mRNA capping, splicing and polyadenylation. Pcf11, an essential component of the mRNA cleavage factor IA, contains a CTD-interaction domain that binds in a phospho-dependent manner to the heptad repeats within the RNA polymerase II CTD. We show here that the phosphorylated CTD exists as a dynamic disordered ensemble in solution and, by induced fit, it assumes a structured conformation when bound to Pcf11. In addition, we detected cis-trans populations for the CTD prolines, and found that only the all-trans form is selected for binding. These data suggest that the recognition of the CTD is regulated by independent site-specific modifications (phosphorylation and proline cis-trans isomerization) and, probably, by the local concentration of suitable binding sites.

Structure of a Mycobacterium tuberculosis NusA–RNA complex

NusA is a key regulator of bacterial transcriptional elongation, pausing, termination and antitermination, yet relatively little is known about the molecular basis of its activity in these fundamental processes. In Mycobacterium tuberculosis, NusA has been shown to bind with high affinity and specificity to BoxB–BoxA–BoxC antitermination sequences within the leader region of the single ribosomal RNA (rRNA) operon. We have determined high‐resolution X‐ray structures of a complex of NusA with two short oligo‐ribonucleotides derived from the BoxC stem–loop motif and have characterised the interaction of NusA with a variety of RNAs derived from the antitermination region. These structures reveal the RNA bound in an extended conformation to a large interacting surface on both KH domains. Combining structural data with observed spectral and calorimetric changes, we now show that NusA binding destabilises secondary structure within rRNA antitermination sequences and propose a model where NusA functions as a chaperone for nascently forming RNA structures.

Crystal structure of the anti-fungal target N-myristoyl transferase

N-myristoyl transferase (NMT) catalyzes the transfer of the fatty acid myristate from myristoyl-CoA to the N-terminal glycine of substrate proteins, and is found only in eukaryotic cells. The enzyme in this study is the 451 amino acid protein produced by Candida albicans, a yeast responsible for the majority of systemic infections in immuno-compromised humans. NMT activity is essential for vegetative growth, and the structure was determined in order to assist in the discovery of a selective inhibitor of NMT which could be developed as an anti-fungal drug. NMT has no sequence homology with other protein sequences and has a novel α/β fold which shows internal twofold symmetry, which may be a result of gene duplication. On one face of the protein there is a long, curved, relatively uncharged groove, at the center of which is a deep pocket. The pocket floor is negatively charged due to the vicinity of the C-terminal carboxylate and a nearby conserved glutamic acid residue, which separates the pocket from a cavity. These observations, considered alongside the positions of residues whose mutation affects substrate binding and activity, suggest that the groove and pocket are the sites of substrate binding and the floor of the pocket is the catalytic center.

Structural basis of lentiviral subversion of a cellular protein degradation pathway

Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits human immunodeficiency virus (HIV)-1 infection of myeloid-lineage cells as well as resting CD4+ T cells by reducing the cellular deoxynucleoside 5′-triphosphate (dNTP) concentration to a level at which the viral reverse transcriptase cannot function. In other lentiviruses, including HIV-2 and related simian immunodeficiency viruses (SIVs), SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation. The molecular mechanism by which these viral proteins are able to usurp the host cell’s ubiquitination machinery to destroy the cell’s protection against these viruses has not been defined. Here we present the crystal structure of a ternary complex of Vpx with the human E3 ligase substrate adaptor DCAF1 and the carboxy-terminal region of human SAMHD1. Vpx is made up of a three-helical bundle stabilized by a zinc finger motif, and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C terminus, making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure reported here provides a molecular description of how a lentiviral accessory protein is able to subvert the cell’s normal protein degradation pathway to inactivate the cellular viral defence system.

Structure of B-MLV capsid amino-terminal domain reveals key features of viral tropism, gag assembly and core formation

The Gag polyprotein is the major structural protein found in all classes of retroviruses. Interactions between Gag molecules control key events at several stages in the cycle of infection. In particular, the capsid (CA) domain of Gag mediates many of the protein-protein interactions that drive retrovirus assembly, maturation and disassembly. Moreover, in murine leukaemia virus (MLV), sequence variation in CA confers N and B tropism that determines susceptibility to the intracellular restriction factors Fv1n and Fv1b. We have determined the structure of the N-terminal domain (NtD) of CA from B-tropic MLV. A comparison of this structure with that of the NtD of CA from N-tropic MLV reveals that although the crystals belong to different space groups, CA monomers are packed with the same P6 hexagonal arrangement. Moreover, interhexamer crystal contacts between residues located at the periphery of the discs are conserved, indicating that switching of tropism does not result in large differences in the backbone conformation, nor does it alter the quaternary arrangement of the disc. We have also examined crystals of the N-tropic MLV CA containing both N- and C-terminal domains. In this case, the NtD hexamer is still present; however, the interhexamer spacing is increased and the conserved interhexamer contacts are absent. Investigation into the effects of mutation of residues that mediate interhexamer contacts reveals that amino acid substitutions at these positions cause severe defects in viral assembly, budding and Gag processing. Based on our crystal structures and mutational analysis, we propose that in MLV, interactions between the NtDs of CA are required for packing of Gag molecules in the early part of immature particle assembly. Moreover, we present a model where proteolytic cleavage at maturation results in migration of CA C-terminal domains into interstitial spaces between NtD hexamers. As a result, NtD-mediated interhexamer contacts present in the immature particle are displaced and the less densely packed lattice with increased hexamer-hexamer spacing characteristic of the viral core is produced.

Structural studies of postentry restriction factors reveal antiparallel dimers that enable avid binding to the HIV-1 capsid lattice.

Significance Retroviral infection of cells can be blocked by the action of the postentry restriction factors. The Trim5α and Fv1 factors do so by targeting the capsid that surrounds the viral core. The nature of the interaction of these factors with the viral assembly is unclear. We show that these factors form antiparallel dimers that display specificity domains spaced to target motifs on the capsid lattice surface. In doing so Fv1 and Trim5α take advantage of the regularly spaced array of binding sites on the capsid surface, generating avidity to aid recognition of retroviral pathogens. Restriction factors (RFs) form important components of host defenses to retroviral infection. The Fv1, Trim5α, and TrimCyp RFs contain N-terminal dimerization and C-terminal specificity domains that target assembled retroviral capsid (CA) proteins enclosing the viral core. However, the molecular detail of the interaction between RFs and their CA targets is unknown. Therefore, we have determined the crystal structure of the B-box and coiled-coil (BCC) region from Trim5α and used small-angle X-ray scattering to examine the solution structure of Trim5α BCC, the dimerization domain of Fv1 (Fv1Ntd), and the hybrid restriction factor Fv1Cyp comprising Fv1NtD fused to the HIV-1 binding protein Cyclophilin A (CypA). These data reveal that coiled-coil regions of Fv1 and Trim5α form extended antiparallel dimers. In Fv1Cyp, two CypA moieties are located at opposing ends, creating a molecule with a dumbbell appearance. In Trim5α, the B-boxes are located at either end of the coiled-coil, held in place by interactions with a helical motif from the L2 region of the opposing monomer. A comparative analysis of Fv1Cyp and CypA binding to a preformed HIV-1 CA lattice reveals how RF dimerization enhances the affinity of interaction through avidity effects. We conclude that the antiparallel organization of the NtD regions of Fv1 and Trim5α dimers correctly positions C-terminal specificity and N-terminal effector domains and facilitates stable binding to adjacent CA hexamers in viral cores.