Biomedical subjects

S C Harrison

Publications and source records attributed to S C Harrison.

At least 19 recordsLinked to original sources

PubMed2001

The stoichiometry of trimeric SIV glycoprotein interaction with CD4 differs from that of anti-envelope antibody Fab fragments.

Human and simian immunodeficiency viruses infect host lymphoid cells by binding CD4 molecules via their gp160 envelope glycoproteins. Biochemical studies on recombinant SIVmac32H (pJ5) envelope ectodomain gp140 precursor protein show that the envelope is a trimer. Using size exclusion chromatography, quantitative amino acid analysis, analytical ultracentrifugation, and CD4-based competition assay, we demonstrate that the stoichiometry of CD4 receptor-oligomeric envelope interaction is 1:1. By contrast, Fab fragments of both neutralizing and non-neutralizing monoclonal antibodies bind at a 3:1 ratio. Thus, despite displaying equivalent CD4 binding sites on each of the three gp140 protomers within an uncleaved trimer, only one site binds the soluble 4-domain human CD4 extracellular segment. The anti-cooperativity and the faster k(off) of gp140 trimer:CD4 versus gp120 monomer:CD4 interaction suggest that CD4-induced conformational change is impeded in the intact envelope. The implications of these findings for immunity against human immunodeficiency virus and simian immunodeficiency virus are discussed.

Amino Acids↗

PubMed2001

Papillomavirus capsid protein expression in Escherichia coli: purification and assembly of HPV11 and HPV16 L1.

The L1 major capsid proteins of human papillomavirus (HPV) types 11 and 16 were purified and analyzed for structural integrity and in vitro self-assembly. Proteins were expressed in Escherichia coli as glutathione-S-transferase-L1 (GST-L1) fusions and purified to near homogeneity as pentamers (equivalent to viral capsomeres), after thrombin cleavage from the GST moiety and removal of tightly associated GroEL protein. Sequences at the amino and carboxy termini contributing to formation of L1 pentamers and to in vitro capsid assembly were identified by deletion analysis. For both HPV11 and HPV16 L1, up to at least ten residues could be deleted from the amino terminus (Delta N10) and 30 residues from the carboxy terminus (Delta C30) without affecting pentamer formation. The HPV16 pentamers assembled into relatively regular, 72-pentamer shells ("virus-like particles" or VLPs) at low pH, with the exception of HPV16 L1 Delta N10, which assembled into a 12-pentamer, T=1 capsid (small VLP) under all conditions tested. The production of large quantities of assembly-competent L1, using the expression and purification protocol described here, has been useful for crystallographic analysis, and will be valuable for studies of virus-receptor interactions and potentially for vaccine design.

Biopolymers↗

PubMed2001

Structure of the reovirus outer capsid and dsRNA-binding protein sigma3 at 1.8 A resolution.

The crystallographically determined structure of the reovirus outer capsid protein sigma3 reveals a two-lobed structure organized around a long central helix. The smaller of the two lobes includes a CCHC zinc-binding site. Residues that vary between strains and serotypes lie mainly on one surface of the protein; residues on the opposite surface are conserved. From a fit of this model to a reconstruction of the whole virion from electron cryomicroscopy, we propose that each sigma3 subunit is positioned with the small lobe anchoring it to the protein mu1 on the surface of the virion, and the large lobe, the site of initial cleavages during entry-related proteolytic disassembly, protruding outwards. The surface containing variable residues faces solvent. The crystallographic asymmetric unit contains two sigma3 subunits, tightly associated as a dimer. One broad surface of the dimer has a positively charged surface patch, which extends across the dyad. In infected cells, sigma3 binds dsRNA and inhibits the interferon response. The location and extent of the positively charged surface patch suggest that the dimer is the RNA-binding form of sigma3.

Amino Acid Sequence↗

PubMed2001

The familiar and the unexpected in structures of icosahedral viruses.

Viruses were the first large macromolecular assemblages to be visualized at high resolution. New virus structures continue to challenge our understanding of specificity in protein-protein "recognition". The evolution of virus structures has been even more opportunistic than previously imagined.

Biological Evolution↗

PubMed2001

Molecular organization of a recombinant subviral particle from tick-borne encephalitis virus.

The tick-borne encephalitis (TBE) flavivirus contains two transmembrane proteins, E and M. Coexpression of E and the M precursor (prM) leads to secretion of recombinant subviral particles (RSPs). In the most common form of these RSPs, analyzed at a 19 A resolution by cryo-electron microscopy (cryo-EM), 60 copies of E pack as dimers in a T = 1 icosahedral surface lattice (outer diameter, 315 A). Fitting the high-resolution structure of a soluble E fragment into the RSP density defines interaction sites between E dimers, positions M relative to E, and allows assignment of transmembrane regions of E and M. Lateral interactions among the glycoproteins stabilize this capsidless particle; similar interactions probably contribute to assembly of virions. The structure suggests a picture for trimer association under fusion-inducing conditions.

Cryoelectron Microscopy↗

PubMed2001

A small domain of CBP/p300 binds diverse proteins: solution structure and functional studies.

The transcriptional coactivators CBP and p300 are critical regulators of metazoan gene expression. They associate with many different DNA-bound transcription factors through small, conserved domains. We have identified a compactly folded 46 residue domain in CBP and p300, the IRF-3 binding domain (IBiD), and we have determined its structure by NMR. It has a helical framework containing an apparently flexible polyglutamine loop that participates in ligand binding. Spectroscopic data indicate that induced folding accompanies association of IBiD with its partners, which exhibit no evident sequence similarities. We demonstrate the significance both in vitro and in vivo of interactions between IBiD and a number of diverse partners. Thus, IBiD is an important contributor to signal integration by CBP and p300.

Amino Acid Sequence↗

PubMed2001

Proteolysis of monomeric recombinant rotavirus VP4 yields an oligomeric VP5* core.

Rotavirus particles are activated for cell entry by trypsin cleavage of the outer capsid spike protein, VP4, into a hemagglutinin, VP8*, and a membrane penetration protein, VP5*. We have purified rhesus rotavirus VP4, expressed in baculovirus-infected insect cells. Purified VP4 is a soluble, elongated monomer, as determined by analytical ultracentrifugation. Trypsin cleaves purified VP4 at a number of sites that are protected on the virion and yields a heterogeneous group of protease-resistant cores of VP5*. The most abundant tryptic VP5* core is trimmed past the N terminus associated with activation for virus entry into cells. Sequential digestion of purified VP4 with chymotrypsin and trypsin generates homogeneous VP8* and VP5* cores (VP8CT and VP5CT, respectively), which have the authentic trypsin cleavages in the activation region. VP8CT is a soluble monomer composed primarily of beta-sheets. VP5CT forms sodium dodecyl sulfate-resistant dimers. These results suggest that trypsinization of rotavirus particles triggers a rearrangement in the VP5* region of VP4 to yield the dimeric spikes observed in icosahedral image reconstructions from electron cryomicroscopy of trypsinized rotavirus virions. The solubility of VP5CT and of trypsinized rotavirus particles suggests that the trypsin-triggered conformational change primes VP4 for a subsequent rearrangement that accomplishes membrane penetration. The domains of VP4 defined by protease analysis contain all mapped neutralizing epitopes, sialic acid binding residues, the heptad repeat region, and the membrane permeabilization region. This biochemical analysis of VP4 provides sequence-specific structural information that complements electron cryomicroscopy data and defines targets and strategies for atomic-resolution structural studies.

Animals↗

PubMed2000

Purified recombinant rotavirus VP7 forms soluble, calcium-dependent trimers.

Rotavirus is a major cause of severe, dehydrating childhood diarrhea. VP7, the rotavirus outer capsid glycoprotein, is a target of protective antibodies and is responsible for the calcium-dependent uncoating of the virus during cell entry. We have purified, characterized, and crystallized recombinant rhesus rotavirus VP7, expressed in insect cells. A critical aspect of the purification is the elution of VP7 from a neutralizing monoclonal antibody column by EDTA. Gel filtration chromatography and equilibrium analytical ultracentrifugation demonstrate that, in the presence of calcium, purified VP7 trimerizes. Trimeric VP7 crystallizes into hexagonal plates. Preliminary X-ray analysis suggests that the crystal packing reproduces the hexagonal component of the icosahedral lattice of VP7 on triple-layered rotavirus particles. These data indicate that the rotavirus outer capsid assembles from calcium-dependent VP7 trimers and that dissociation of these trimers is the biochemical basis for EDTA-induced rotavirus uncoating and loss of VP7 neutralizing epitopes.

Animals↗

PubMed2000

Expression, purification, and characterization of gp160e, the soluble, trimeric ectodomain of the simian immunodeficiency virus envelope glycoprotein, gp160.

The envelope glycoprotein, gp160, of simian immunodeficiency virus (SIV) shares approximately 25% sequence identity with gp160 from the human immunodeficiency virus, type I, indicating a close structural similarity. As a result of binding to cell surface CD4 and co-receptor (e.g. CCR5 and CXCR4), both SIV and human immunodeficiency virus gp160 mediate viral entry by membrane fusion. We report here the characterization of gp160e, the soluble ectodomain of SIV gp160. The ectodomain has been expressed in both insect cells and Chinese hamster ovary (CHO)-Lec3.2.8.1 cells, deficient in enzymes necessary for synthesizing complex oligosaccharides. Both the primary and a secondary proteolytic cleavage sites between the gp120 and gp41 subunits of gp160 were mutated to prevent cleavage and shedding of gp120. The purified, soluble glycoprotein is shown to be trimeric by chemical cross-linking, gel filtration chromatography, and analytical ultracentrifugation. It forms soluble, tight complexes with soluble CD4 and a number of Fab fragments from neutralizing monoclonal antibodies. Soluble complexes were also produced of enzymatically deglycosylated gp160e and of gp160e variants with deletions in the variable segments.

Animals↗

PubMed2000

Structure of PAK1 in an autoinhibited conformation reveals a multistage activation switch.

The p21-activated kinases (PAKs), stimulated by binding with GTP-liganded forms of Cdc42 or Rac, modulate cytoskeletal actin assembly and activate MAP-kinase pathways. The 2.3 A resolution crystal structure of a complex between the N-terminal autoregulatory fragment and the C-terminal kinase domain of PAK1 shows that GTPase binding will trigger a series of conformational changes, beginning with disruption of a PAK1 dimer and ending with rearrangement of the kinase active site into a catalytically competent state. An inhibitory switch (IS) domain, which overlaps the GTPase binding region of PAK1, positions a polypeptide segment across the kinase cleft. GTPase binding will refold part of the IS domain and unfold the rest. A related switch has been seen in the Wiskott-Aldrich syndrome protein (WASP).

Amino Acid Sequence↗

PubMed2000

Structure of the reovirus core at 3.6 A resolution.

The reovirus core is an assembly with a relative molecular mass of 52 million that synthesizes, modifies and exports viral messenger RNA. Analysis of its structure by X-ray crystallography shows that there are alternative, specific and completely non-equivalent contacts made by several surfaces of two of its proteins; that the RNA capping and export apparatus is a hollow cylinder, which probably sequesters its substrate to ensure completion of the capping reactions; that the genomic double-stranded RNA is coiled into concentric layers within the particle; and that there is a protein shell that appears to be common to all groups of double-stranded RNA viruses.

Capsid↗

PubMed2000

Peptide-in-groove interactions link target proteins to the beta-propeller of clathrin.

The "WD40" domain is a widespread recognition module for linking partner proteins in intracellular networks of signaling and sorting. The clathrin amino-terminal domain, which directs incorporation of cargo into coated pits, is a beta-propeller closely related in structure to WD40 modules. The crystallographically determined structures of complexes of the clathrin-terminal domain with peptides derived from two different cargo adaptors, beta-arrestin 2 and the beta-subunit of the AP-3 complex, reveal strikingly similar peptide-in-groove interactions. The two peptides in our structures contain related, five-residue motifs, which form the core of their contact with clathrin. A number of other proteins involved in endocytosis have similar "clathrin-box" motifs, and it therefore is likely that they all bind the terminal domain in the same way. We propose that a peptide-in-groove interaction is an important general mode by which beta-propellers recognize specific target proteins.

Adaptor Proteins, Vesicular Transport↗

PubMed2000

Identification of the guanylyltransferase region and active site in reovirus mRNA capping protein lambda2.

The 144-kDa lambda2 protein of mammalian reovirus catalyzes a number of enzymatic activities in the capping of reovirus mRNA, including the transfer of GMP from GTP to the 5' end of the 5'-diphosphorylated nascent transcript. This reaction proceeds through a covalently autoguanylylated lambda2-GMP intermediate. The smaller size of RNA capping guanylyltransferases from other organisms suggested that the lambda2-associated guanylyltransferase would be only a part of this protein. Limited proteinase K digestion of baculovirus-expressed lambda2 was used to generate an amino-terminal M(r) 42,000 fragment that appears to be both necessary and sufficient for guanylyltransferase activity. Although lysine 226 was identified by previous biochemical studies as the active-site residue that forms a phosphoamide bond with GMP in autoguanylylated lambda2, mutation of lysine 226 to alanine caused only a partial reduction in guanylyltransferase activity at the autoguanylylation step. Alanine substitution for other lysines within the amino-terminal region of lambda2 identified lysine 190 as necessary for autoguanylylation and lysine 171 as an important contributor to autoguanylylation. A novel active-site motif is proposed for the RNA guanylyltransferases of mammalian reoviruses and other Reoviridae members.

Animals↗

PubMed2000

The structure of an HIV-1 specific cell entry inhibitor in complex with the HIV-1 gp41 trimeric core.

The three-dimensional structure of the complex between an HIV-1 cell-entry inhibitor selected from screening a combinatorial library of non-natural building blocks and the central, trimeric, coiled-coil core of HIV-1 gp41 has been determined by X-ray crystallography. The biased combinatorial library was designed to identify ligands binding in nonpolar pockets on the surface of the coiled-coil core of gp41. The crystal structure shows that the non-peptide moiety of the inhibitor binds to the targeted cavity in two different binding modes. This result suggests a strategy for increasing inhibitor potency by use of a second-generation combinatorial library designed to give simultaneous occupancy of both binding sites.

Anti-HIV Agents↗

PubMed2000

Trapping of a catalytic HIV reverse transcriptase*template:primer complex through a disulfide bond.

BACKGROUND: HIV-1 reverse transcriptase (RT) is a major target for the treatment of acquired immunodeficiency syndrome (AIDS). Resistance mutations in RT compromise treatment, however. Efforts to understand the enzymatic mechanism of RT and the basis for mutational resistance to anti-RT drugs have been hampered by the failure to crystallize a catalytically informative RT-substrate complex. RESULTS: We present here experiments that allow us to understand the reason for the failure to crystallize such a complex. Based on this understanding, we have devised a new approach for using a combinatorial disulfide cross-linking strategy to trap a catalytic RT*template:primer*dNTP ternary complex, thereby enabling the growth of co-crystals suitable for high-resolution structural analysis. The crystals contain a fully assembled active site poised for catalysis. The cross-link itself appears to be conformationally mobile, and the surrounding region is undistorted, suggesting that the cross-link is a structurally passive device that merely acts to prevent dissociation of the catalytic complex. CONCLUSIONS: The new strategy discussed here has resulted in the crystallization and structure determination of a catalytically relevant RT*template:primer*dNTP complex. The structure has allowed us to analyze possible causes of drug resistance at the molecular level. This information will assist efforts to develop new classes of nucleoside analog inhibitors, which might help circumvent current resistance profiles. The covalent trapping strategy described here may be useful with other protein-DNA complexes that have been refractory to structural analysis.

Catalysis↗

PubMed2000

Structure of small virus-like particles assembled from the L1 protein of human papillomavirus 16.

The papillomavirus major late protein, L1, forms the pentameric assembly unit of the viral shell. Recombinant HPV16 L1 pentamers assemble in vitro into capsid-like structures, and truncation of ten N-terminal residues leads to a homogeneous preparation of 12-pentamer, icosahedral particles. X-ray crystallographic analysis of these particles at 3.5 A resolution shows that L1 closely resembles VP1 from polyomaviruses. Surface loops contain the sites of sequence variation among HPV types and the locations of dominant neutralizing epitopes. The ease with which small virus-like particles may be obtained from L1 expressed in E. coli makes them attractive candidate components of a papillomavirus vaccine. Their crystal structure also provides a starting point for future vaccine design.

Amino Acid Sequence↗

PubMed2000

Is bowel preparation useful before radiography of the renal tract in patients with spinal cord injury?

OBJECTIVES: To assess, in a blinded study, the usefulness of bowel preparation in improving the quality of radiography of the renal tract in patients with spinal cord injury (SCI). PATIENTS AND METHODS: Plain abdominal radiographs of 56 patients with SCI were selected; 24 of the patients had received bowel preparation and 32 had not. The films were independently assessed by one radiologist and one urologist unaware of the treatment and identity of the patients. Each film was divided into five regions of interest and scores of 1-4 (1 for least and 4 for best visibility) assigned to each area. In films with a low aggregate visibility score (</= 12), the cause of poor visibility was assessed in relation to faecal residue and gas. RESULTS: The difference between the overall mean visibility score for bowel-prepared and unprepared patients was not statistically significant. In films with poor visibility, gas shadows predominated over bowel shadows in the bowel-prepared group, although this trend was not statistically significant. Of the five areas, the bladder was scored as being the most clearly seen in both groups. CONCLUSIONS: These findings suggest that bowel preparation does not increase the diagnostic value of radiographs of the renal tract in patients with SCI.

Cathartics↗

PubMed1999

Crystal structure of the ectodomain of human transferrin receptor.

The transferrin receptor (TfR) undergoes multiple rounds of clathrin-mediated endocytosis and reemergence at the cell surface, importing iron-loaded transferrin (Tf) and recycling apotransferrin after discharge of iron in the endosome. The crystal structure of the dimeric ectodomain of the human TfR, determined here to 3.2 angstroms resolution, reveals a three-domain subunit. One domain closely resembles carboxy- and aminopeptidases, and features of membrane glutamate carboxypeptidase can be deduced from the TfR structure. A model is proposed for Tf binding to the receptor.

Amino Acid Sequence↗