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S C Harrison

Publications and source records attributed to S C Harrison.

At least 55 records · Page 3Linked to original sources
PubMed1997

High-resolution structure of a polyomavirus VP1-oligosaccharide complex: implications for assembly and receptor binding.

The crystal structure of a recombinant polyomavirus VP1 pentamer (residues 32-320) in complex with a branched disialylated hexasaccharide receptor fragment has been determined at 1.9 A resolution. The result extends our understanding of oligosaccharide receptor recognition. It also suggests a mechanism for enhancing the fidelity of virus assembly. We have previously described the structure of the complete polyomavirus particle complexed with this receptor fragment at 3.65 A. The model presented here offers a much more refined view of the interactions that determine carbohydrate recognition and allows us to assign additional specific contacts, in particular those involving the (alpha2,6)-linked, branching sialic acid. The structure of the unliganded VP1 pentamer, determined independently, shows that the oligosaccharide fits into a preformed groove and induces no measurable structural rearrangements. A comparison with assembled VP1 in the virus capsid reveals a rearrangement of residues 32-45 at the base of the pentamer. This segment may help prevent the formation of incorrectly assembled particles by reducing the likelihood that the C-terminal arm will fold back into its pentamer of origin.

Amino Acid Sequence↗
PubMed1997

Atomic structure of the ectodomain from HIV-1 gp41.

Fusion of viral and cellular membranes by the envelope glycoprotein gp120/gp41 effects entry of HIV-1 into the cell. The precursor, gp160, is cleaved post-translationally into gp120 and gp41 which remain non-covalently associated. Binding to both CD4 and a co-receptor leads to the conformational changes in gp120/gp41 needed for membrane fusion. We used X-ray crystallography to determine the structure of the protease-resistant part of a gp41 ectodomain solubilized with a trimeric GCN4 coiled coil in place of the amino-terminal fusion peptide. The core of the molecule is found to be an extended, triple-stranded alpha-helical coiled coil with the amino terminus at its tip. A carboxy-terminal alpha-helix packs in the reverse direction against the outside of the coiled coil, placing the amino and carboxy termini near each other at one end of the long rod. These features, and the existence of a similar reversal of chain direction in the fusion pH-induced conformation of influenza virus HA2 and in the transmembrane subunit of Moloney murine leukaemia virus (Fig. 1a-d), suggest a common mechanism for initiating fusion.

Amino Acid Sequence↗
PubMed1997

Crystal structure of ICAM-2 reveals a distinctive integrin recognition surface.

Recognition by integrin proteins on the cell surface regulates the adhesive interactions between cells and their surroundings. The structure of the 'I' domain that is found in some but not all integrins, has been determined. However, the only integrin ligands for which structures are known, namely fibronectin and VCAM-1, are recognized by integrins that lack I domains. The intercellular adhesion molecules ICAM-1, 2 and 3 are, like VCAM-1, members of the immunoglobulin superfamily (IgSF), but they are recognized by an I domain-containing integrin, lymphocyte-function-associated antigen 1 (LFA-1, or CD11a/CD18). Here we present the crystal structure of the extracellular region of ICAM-2. The glutamic acid residue at position 37 is critical for LFA-1 binding and is proposed to coordinate the Mg2+ ion in the I domain; this Glu 37 is surrounded by a relatively flat recognition surface and lies in a beta-strand, whereas the critical aspartic acid residue in VCAM-1 and fibronectin lie in protruding loops. This finding suggests that there are differences in the architecture of recognition sites between integrins that contain or lack I domains. A bend between domains 1 and 2 of ICAM-2 and a tripod-like arrangement of N-linked glycans in the membrane-proximal region of domain 2 may be important for presenting the recognition surface to LFA-1. A model of ICAM-1 based on the ICAM-2 structure provides a framework for understanding its recognition by pathogens.

Amino Acid Sequence↗
PubMed1997

Three-dimensional structure of the tyrosine kinase c-Src.

The structure of a large fragment of the c-Src tyrosine kinase, comprising the regulatory and kinase domains and the carboxy-terminal tall, has been determined at 1.7 A resolution in a closed, inactive state. Interactions among domains, stabilized by binding of the phosphorylated tail to the SH2 domain, lock the molecule in a conformation that simultaneously disrupts the kinase active site and sequesters the binding surfaces of the SH2 and SH3 domains. The structure shows how appropriate cellular signals, or transforming mutations in v-Src, could break these interactions to produce an open, active kinase.

Amino Acid Sequence↗
PubMed1996

Crystal structure of the breakpoint cluster region-homology domain from phosphoinositide 3-kinase p85 alpha subunit.

Proteins such as the product of the break-point cluster region, chimaerin, and the Src homology 3-binding protein 3BP1, are GTPase activating proteins (GAPs) for members of the Rho subfamily of small GTP-binding proteins (G proteins or GTPases). A 200-residue region, named the breakpoint cluster region-homology (BH) domain, is responsible for the GAP activity. We describe here the crystal structure of the BH domain from the p85 subunit of phosphatidylinositol 3-kinase at 2.0 A resolution. The domain is composed of seven helices, having a previously unobserved arrangement. A core of four helices contains most residues that are conserved in the BH family. Their packing suggests the location of a G-protein binding site. This structure of a GAP-like domain for small GTP-binding proteins provides a framework for analyzing the function of this class of molecules.

Amino Acid Sequence↗
PubMed1996

Phosphorylated T cell receptor zeta-chain and ZAP70 tandem SH2 domains form a 1:3 complex in vitro.

The zeta polypeptide is part of the T cell antigen receptor (TCR). The zeta-chain contributes to efficient cell-surface expression of the TCR and accounts for part of its signal transduction capability. TCR recognition triggers a complex set of events that result in cellular activation. The protein tyrosine kinase (PTK) Lck phosphorylates the zeta-chain, which in turn associates with another PTK, ZAP70, and stimulates its phosphorylation activity. Here we report the expression of the intracellular part of the zeta-chain and its biochemical characterization. The recombinant protein does not dimerize by itself in solution. Circular-dichroic analysis reveals a random coil conformation. zeta, phosphorylated using recombinant Lck, associates with recombinant ZAP70 tandem-SH2 domains. All three T cell activation motifs in zeta bind ZAP70 tandem-SH2 domains in vitro, forming a 1:3 complex. This result extends the picture, derived from earlier studies, of a mechanism for signal amplification.

Binding Sites↗
PubMed1996

DNA transport by a type II topoisomerase: direct evidence for a two-gate mechanism.

Recent biochemical and crystallographic results suggest that a type II DNA topoisomerase acts as an ATP-modulated clamp with two sets of jaws at opposite ends: a DNA-bound enzyme can admit a second DNA through one set of jaws; upon binding ATP, this DNA is passed through an enzyme-mediated opening in the first DNA and expelled from the enzyme through the other set of jaws. Experiments based on the introduction of reversible disulfide links across one dimer interface of yeast DNA topoisomerase II have confirmed this mechanism. The second DNA is found to enter the enzyme through the gate formed by the N-terminal parts of the enzyme and leave it through the gate close to the C termini.

Adenosine Triphosphate↗
PubMed1996

Structure determination of simian virus 40 and murine polyomavirus by a combination of 30-fold and 5-fold electron-density averaging.

BACKGROUND: Simian virus 40 (SV40) and murine polyomavirus (polyoma) are non-enveloped DNA tumor viruses. Their structurally similar capsids, about 500 degrees in diameter, are formed by 72 pentamers of the major coat protein VP1. RESULTS: We describe in this paper the structure determination of SV40 and polyoma at 3.8 degree resolution, focusing particularly on methodological issues, and on a comparison of the overall molecular organization in the two related virus particles. Initial phases for SV40 were obtained by single isomorphous replacement at 6.5 degree. Phases were refined and the resolution extended to 3.8 degree by a combination of strict 5-fold and partial 30-fold electron-density averaging. The structure of polyoma was subsequently determined by systematically translating and rotating the individual VP1 pentamers, in order to find the maximum correlation between calculated and observed structure factors. The resolution was then extended to 3.8 degree, also by phase refinement through electron-density averaging. CONCLUSION: The strategies for density averaging and for molecular replacement, used to determine the SV40 and polyoma structures, are likely to be generally useful. The individual building blocks, the VP1 pentamers, are essentially identical in both cases, as are the local details of their interactions with neighboring pentamers. Nevertheless, the arrangement of the pentamers with respect to each other is somewhat different in the two viruses. Whereas SV40 is almost spherical, with all pentamers at identical radii, the pentamers in polyoma that lie on icosahedral fivefold axes are displaced outward by about 5 degree.

Capsid↗
PubMed1996

The structure of simian virus 40 refined at 3.1 A resolution.

BACKGROUND: The structure of simian virus 40 (SV40), previously determined at 3.8 degree resolution, shows how its pentameric VP1 assembly units are tied together by extended C-terminal arms. In order to define more precisely the possible assembly mechanisms, we have refined the structure at 3.1 degree resolution. RESULTS: New data from a high-intensity synchrotron source have been used for phase extension by electron-density averaging and refinement, exploiting only the strict 5-fold non-crystallographic symmetry for the real-space averaging steps. The accurate model enables us to study important structural features of the virus particle in detail. The remarkably invariant core of the VP1 pentamer bears the docking sites for the C-terminal arms from other pentamers. These contacts are the principal way in which pentameric assembly units are linked together in the capsid. Only at the interface between five-coordinated and six-coordinated pentamers do the pentamer cores appear to interact strongly. There are two cation-binding sites per VP1 monomer, seen in a soaking experiment with gadolinium nitrate. These sites are quite close to each other at the interfaces between pentamers. CONCLUSION: We propose that the contact between five-coordinated and six-coordinated pentamers may help to generate a six-pentamer nucleus, with which further pentamers can assemble to generate the complete particle. Calcium ions probably stabilize the structure of the assembled particle, rather than direct its assembly.

Amino Acid Sequence↗
PubMed1996

Crystal structures of murine polyomavirus in complex with straight-chain and branched-chain sialyloligosaccharide receptor fragments.

BACKGROUND: Murine polyomavirus recognizes (alpha2,3)-linked alpha-5-N-acetylneuraminic acid (sialic acid) on the surface of susceptible cells. While all strains bind to straight-chain receptors terminating in (alpha2,3)-linked sialic acid, some strains also bind to branched oligosaccharides that carry a second, (alpha2,6)-linked sialic acid. The ability to bind to these branched-chain receptors correlates with a single amino acid mutation at position 91 on the outer surface of the major capsid protein, VP1, and with a significant decrease in tumorigenicity. RESULTS: We have determined the structures of polyomavirus strain P16, which bears a glycine at position 91, in complex with model compounds for both straight-chain and branched-chain sialoglycoconjugates. The structures have been refined to a resolution of 3.65 degree. The ligands bind to a shallow groove on the surface of VP1. The sialic acid-(alpha2,3)-galactose moiety, which is common to both compounds, has specific and identical contacts. The additional (alpha2,6)-linked sialic acid moiety of the branched-chain receptor fragment fits into a surface pocket, but it has high thermal factors and does not form hydrogen bonds to groups on VP1. Data collected from crystals soaked at different oligosaccharide concentrations establish that both receptor fragments have similar, low affinities (dissociation constants in the range 5-10 mM) for the P16 virus, consistent with the interactions seen in the two complexes. CONCLUSION: The oligosaccharide-binding groove is complementary to the shape of the bound glycan, but there are relatively few hydrogen bonds between glycan and protein. Thus, the nature of the glycosidic linkages appears to be the principal determinant of specificity, rather than the position of particular hydroxyl groups. The low receptor affinity may be important for avoiding inhibition of viral release by retention on surface receptors of infected cells. Evidence suggests that strains with still greater pathogenicity are likely to have even weaker affinity.

Amino Acid Sequence↗
PubMed1996

Structure and mechanism of DNA topoisomerase II.

The crystal structure of a large fragment of yeast type II DNA topoisomerase reveals a heart-shaped dimeric protein with a large central hole. It provides a molecular model of the enzyme as an ATP-modulated clamp with two sets of jaws at opposite ends, connected by multiple joints. An enzyme with bound DNA can admit a second DNA duplex through one set of jaws, transport it through the cleaved first duplex, and expel it through the other set of jaws.

Adenosine Triphosphatases↗
PubMed1996

Spatial constraints on the recognition of phosphoproteins by the tandem SH2 domains of the phosphatase SH-PTP2.

The domain organization of many signalling proteins facilitates a segregation of binding, catalytic and regulatory functions. The mammalian SH2 domain protein tyrosine phosphatases (PTPs) contain tandem SH2 domains and a single carboxy-terminal catalytic domain. SH-PTP1 (PTP1C, HCP) and SH-PTP2 (Syp, PTP2C, PTP1D) function downstream from tyrosine kinase-linked insulin, growth factor, cytokine and antigen receptors. As well as directing subcellular localization by binding to receptors and their substrates, the two SH2 domains of these PTPs function together to regulate catalysis. Here we report the structure of the tandem SH2 domains of SH-PTP2 in complex with monophosphopeptides. A fixed relative orientation of the two domains, stabilized by a disulphide bond and a small hydrophobic patch within the interface, separates the peptide binding sites by approximately 40 A. The defined orientation of the SH2 domains in the structure, and data showing that peptide orientation and spacing between binding sites is critical for enzymatic activation, suggest that spatial constraints are important in this multidomain protein-protein interaction.

Amino Acid Sequence↗
PubMed1996

Crystal structure of the PI 3-kinase p85 amino-terminal SH2 domain and its phosphopeptide complexes.

Crystal structures of the amino-terminal SH2 domain of the p85alpha subunit of phosphatidylinositol (PI) 3-kinase, alone and in complex with phosphopeptides bearing pTyr-Met/Val-Xaa-Met motifs, show that phosphopeptides bind in the two-pronged manner seen in high-affinity Lck and Src SH2 complexes, with conserved interactions between the domain and the peptide segment from phosphotyrosine to Met+3. Peptide binding requires the rearrangement of a tyrosyl side chain in the BG loop to create the hydrophobic Met+3 binding pocket. The structures suggest a mechanism for the biological specificity exhibited by PI 3-kinase in its interactions with phosphoprotein partners.

Amino Acid Sequence↗
PubMed1996

Retrovirus envelope domain at 1.7 angstrom resolution.

We report the crystal structure of an extraviral segment of a retrovirus envelope protein, the Moloney murine leukemia virus (MoMuLV) transmembrane (TM) subunit. This segment, which comprises a region of the MoMuLV TM protein analogous to that contained within the X-ray crystal structure of low-pH converted influenza hemagglutinin, contains a trimeric coiled coil, with a hydrophobic cluster at its base and a strand that packs in an antiparallel orientation against the coiled coil. This structure gives the first high-resolution insight into the retrovirus surface and serves as a model for a wide range of viral fusion proteins; key residues in this structure are conserved among C- and D-type retroviruses and the filovirus ebola.

Amino Acid Sequence↗
PubMed1996

DNA sequence preferences of GAL4 and PPR1: how a subset of Zn2 Cys6 binuclear cluster proteins recognizes DNA.

Biophysical and genetic experiments have defined how the Saccharomyces cerevisiae protein GAL4 and a subset of related proteins recognize specific DNA sequences. We assessed DNA sequence preferences of GAL4 and a related protein, PPR1, in an in vitro DNA binding assay. For GAL4, the palindromic CGG triplets at the ends of the 17-bp recognition site are essential for tight binding, whereas the identities of the internal 11 bp are much less important, results consistent with the GAL4-DNA crystal structure. Small reductions in affinity due to mutations at the center-most 5 bp are consistent with the idea that an observed constriction in the minor groove in the crystalline GAL4-DNA complex is sequence dependent. The crystal structure suggests that this sequence dependence is due to phosphate contacts mediated by arginine 51, as part of a network of hydrogen bonds. Here we show that the mutant protein GAL4(1-100)R51A fails to discriminate sites with alterations in the center of the site from the wild-type site. PPR1, a relative of GAL4, also recognizes palindromic CGG triplets at the ends of its 12-bp recognition sequence. The identities of the internal 6 bp do not influence the binding of PPR1. We also show that the PPR1 site consists of a 12-bp duplex rather than 16 bp as reported previously: the two T residues immediately 5' to the CGG sequence in each half site, although highly conserved, are not important for binding by PPR1. Thus, GAL4 and PPR1 share common CGG half sites, but they prefer DNA sequences with the palindromic CGG separated by the appropriate number of base pairs, 11 for GAL4 and 6 for PPR1.

Amino Acid Sequence↗