Biomedical subjects

D C Wiley

Publications and source records attributed to D C Wiley.

At least 73 records · Page 4Linked to original sources

PubMed1997

Structure of the inhibitory receptor for human natural killer cells resembles haematopoietic receptors.

Abnormal cells deficient in class I major histocompatibility complex (MHC) expression are lysed by a class of lymphocytes called natural killer (NK) cells. This lysis provides a defence against pathogens and tumour cells that downregulate MHC expression to avoid an MHC-restricted, T-cell immune response. Normal cells escape lysis because their MHC molecules are recognized by NK-cell inhibitory receptors, which inhibit lysis. Several such inhibitory receptor families have been described in humans and mice. In the human killer-cell inhibitory receptor family, individual p58 members are specific for a subset of class I human leukocyte antigen (HLA)-C molecules. The human p58 natural killer-cell inhibitory receptor clone 42 recognizes HLA-Cw4, -Cw2 and -Cw6, but not HLA-Cw3, -Cw2, -Cw7 or -Cw8, which are recognized by p58 killer-cell inhibitor receptor clone 43. We have determined the X-ray structure of the p58 NK-cell inhibitory receptor clone 42 at 1.7-A resolution. The structure has tandem immunoglobulin-like domains positioned at an acute, 60-degree angle. Loops on the outside of the elbow between the domains form a binding site projected away from the NK-cell surface. The topology of the domains and their arrangement relative to each other reveal a relationship to the haematopoietic receptor family, with implications for the signalling mechanism in NK cells.

Binding Sites↗

PubMed1997

Adaptation of egg-grown and transfectant influenza viruses for growth in mammalian cells: selection of hemagglutinin mutants with elevated pH of membrane fusion.

A series of eight transfectant influenza viruses was generated by reverse genetics for studies of the palmitylated cysteine residues in the cytoplasmic tail of the hemagglutinin glycoprotein (HA). Following amplification of these viruses in MDCK cells we found that all had developed an elevated pH of membrane fusion--an unexpected result since previous mutant HA expression studies had shown that substitutions of the cysteine residues had no effect on fusion properties. Sequence analyses revealed that each of the viruses had at least one additional mutation in the ectodomain of HA which was responsible for the increase in fusion pH. Similarly, when we passaged egg-grown wild-type X-31 virus in three different lines of MDCK cells or in MDBK cells, high pH fusion mutants were selected within a few passages in every case. The locations of the substitutions in the HA structure are in or near the "fusion peptide" or at subunit interfaces throughout the length of the trimer--reminiscent of the changes selected in earlier studies on amantadine resistance. The observation that passage of certain viruses in mammalian cells can result in the selection of mutants with elevated fusion pH has potential implications both for reverse genetic experiments and, perhaps more importantly, for the choice of substrates for propagation of vaccine viruses.

Adaptation, Physiological↗

PubMed1997

Assembly of a rod-shaped chimera of a trimeric GCN4 zipper and the HIV-1 gp41 ectodomain expressed in Escherichia coli.

The HIV-1 envelope subunit gp41 plays a role in viral entry by initiating fusion of the viral and cellular membranes. A chimeric molecule was constructed centered on the ectodomain of gp41 without the fusion peptide, with a trimeric isoleucine zipper derived from GCN4 (pIIGCN4) on the N terminus and part of the trimeric coiled coil of the influenza virus hemagglutinin (HA) HA2 on the C terminus. The chimera pII-41-HA was overexpressed as inclusion bodies in bacteria and refolded to soluble aggregates that became monodisperse after treatment with protease. Either trypsin or proteinase K, used previously to define a protease-resistant core of recombinant gp41 [Lu, M., Blacklow, S. C. & Kim, P. S. (1995) Nat. Struct. Biol. 2, 1075-1082], removed about 20-30 residues from the center of gp41 and all or most of the HA2 segment. Evidence is presented that the resulting soluble chimera, retaining the pIIGCN4 coiled coil at the N terminus, is an oligomeric highly alpha-helical rod about 130 A long that crystallizes. The chimeric molecule is recognized by the Fab fragments of mAbs specific for folded gp41. A similar chimera was assembled from the two halves of the molecule expressed separately in different bacteria and refolded together. Crystals from the smallest chimera diffract x-rays to 2.6-A resolution.

Antibodies↗

PubMed1997

Binding of the influenza A virus to cell-surface receptors: structures of five hemagglutinin-sialyloligosaccharide complexes determined by X-ray crystallography.

The structures of five complexes of the X-31 influenza A (H3N2) virus hemagglutinin with sialyloligosaccharide receptor analogs have been determined from 2.5 to 2.8 A resolution by X-ray crystallography. There is well-defined electron density for three to five saccharides in all five complexes and a striking conformational difference between two linear pentasaccharides with the same composition but different linkage [alpha(2-->6) or alpha(2-->3)] at the terminal sialic acid. The bound position of the terminal sialic acid (NeuAc) is the same in all five complexes and is identical to that reported previously from the study of mono- and trisaccharides. The two oligosaccharides with NeuAc alpha(2-->6)Gal linkages and GlcNAc at the third position have a folded conformation with the GlcNAc doubled back to contact the sialic acid. The pentasaccharide with a terminal NeuAc alpha(2-->3)Gal linkage and GlcNAc at the third position has an extended (not folded) conformation and exits from the opposite side of the binding site than the alpha(2-->6)-linked molecule of the same composition. The difference between the conformation of the pentasaccharide with a 2,6 linkage and the trisaccharide 2,6-sialyllactose suggests that 2,6-sialyllactose is not, as previously believed, an appropriate analog of natural influenza A virus receptors. The oligosaccharides studied are NeuAc alpha(2-->3)Gal beta(1-->4)Glc, NeuAc alpha(2-->6)Gal beta(1-->4)Glc, NeuAc alpha(2-->3)Gal beta(1-->3)GlcNAc beta(1-->3)Gal beta(1-->4)Glc, NeuAc alpha(2-->6)Gal beta(1-->4)GlcNAc beta(1-->3)Gal beta(1-->4)Glc, and [NeuAc alpha(2-->6)Gal beta(1-->4)GlcNAc]2 beta(1-->3/6)Gal-beta-O-(CH2)5-COOCH3.

Carbohydrate 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

X-ray crystal structure of HLA-DR4 (DRA0101, DRB10401) complexed with a peptide from human collagen II.

Genetic predisposition to rheumatoid arthritis (RA) is linked to the MHC class II allele HLA-DR4. The charge of the amino acid at DRbeta71 in the peptide-binding site appears to be critical in discriminating DR molecules linked to increased disease susceptibility. We have determined the 2.5 A x-ray structure of the DR4 molecule with the strongest linkage to RA (DRB1*0401) complexed with a human collagen II peptide. Details of a predicted salt bridge between lysine DRbeta71 and aspartic acid at the P4 peptide position suggest how it may participate in both antigen binding and TCR activation. A model is proposed for the DR4 recognition of collagen II (261-273), an antigen immunodominant in human-transgenic mouse models of RA.

Alleles↗

PubMed1997

Using the Youth Risk Behavior Survey to compare risk behaviors of Texas High School and college students.

This study used the Youth Risk Behavior Survey (YRBS) to assess selected health behaviors of Texas high school and college students. The YRBS was administered during 1993 in paper and pencil form to 6,015 high school students representing 329 classrooms from 78 school districts. A total of 1,408 college students representing 23 college and universities were surveyed by telephone in 1993 using a modified version of YRBS. Texas college students reported a higher percentage who had experienced sexual intercourse (82% versus 55.4%), but Texas high school students reported a younger age of first sexual intercourse. High school students also initiated alcohol consumption at a younger age, although college students were more likely to binge drink (33.5% versus 31%). Regular cigarette use also was higher among college students (25.4% versus 19.3%), but was initiated at a younger age by high school students. Study results indicate that health education programs must begin much earlier than during the high school years. Due to early initiation of negative health behaviors, emphasis must be placed on abstinence and risk-reduction techniques for both populations.

Adolescent↗

PubMed1996

Assembly, specific binding, and crystallization of a human TCR-alphabeta with an antigenic Tax peptide from human T lymphotropic virus type 1 and the class I MHC molecule HLA-A2.

T lymphocytes use TCR-alphabeta to bind and to recognize complexes of antigenic peptides bound to MHC proteins located at the surface of APCs. We have assembled and crystallized this intercellular complex of TCR/peptide/MHC from soluble human TCR-alphabeta and soluble peptide/HLA-A2 complexes. The soluble TCR-alphabeta binds specifically to its in vivo ligand, the complex of HLA-A2, and a peptide from the Tax protein of human T lymphotropic virus type 1. The soluble TCR also binds in vitro to an altered peptide ligand, which appears to be a partial agonist in T cell assays as determined by its ability to elicit different cytolytic and lymphokine secretion responses. Heterodimerization and the antigenic specificity of the TCR do not require its interchain disulfide bond, transmembrane segments, or glycosylations. Crystals of the TCR/peptide/HLA-A2 complex diffract x-rays, providing the means to study in atomic detail the mechanism of Ag-specific cell-cell recognition between T cells and target cells.

Amino Acid Sequence↗

PubMed1996

Structure of the complex between human T-cell receptor, viral peptide and HLA-A2.

Recognition by a T-cell antigen receptor (TCR) of peptide complexed with a major histocompatibility complex (MHC) molecule occurs through variable loops in the TCR structure which bury almost all the available peptide and a much larger area of the MHC molecule. The TCR fits diagonally across the MHC peptide-binding site in a surface feature common to all class I and class II MHC molecules, providing evidence that the nature of binding is general. A broadly applicable binding mode has implications for the mechanism of repertoire selection and the magnitude of alloreactions.

Crystallography, X-Ray↗

PubMed1996

Studies using double mutants of the conformational transitions in influenza hemagglutinin required for its membrane fusion activity.

Amino acid substitutions widely distributed throughout the influenza hemagglutinin (HA) influence the pH of its membrane fusion activity. We have combined a number of these substitutions in double mutants and determined the effects on the pH of fusion and on the pH at which the refolding of HA required for fusion occurs. By analyzing combinations of mutations in three regions of the metastable neutral-pH HA that are rearranged at fusion pH we obtain evidence for both additive and nonadditive effects and for an apparent order of dominance in the effects of amino acid substitutions in particular regions on the pH of fusion. We conclude that there are at least three components in the structural transition required for membrane fusion activity and consider possible pathways for the transition in relation to the known differences between neutral and fusion pH HA structures.

Aspartic Acid↗

PubMed1996

Covalent HLA-B27/peptide complex induced by specific recognition of an aziridine mimic of arginine.

The class I major histocompatibility complex (MHC) glycoprotein HLA-B27 binds short peptides containing arginine at peptide position 2 (P2). The HLA-B27/peptide complex is recognized by T cells both as part of the development of the repertoire of T cells in the cellular immune system and during activation of cytotoxic T cells. Based on the three-dimensional structure of HLA-B27, we have synthesized a ligand with an aziridine-containing side chain designed to mimic arginine and to bind covalently in the arginine-specific P2 pocket of HLA-B27. Using tryptic digestion followed by mass spectrometry and amino acid sequencing, the aziridine-containing ligand is shown to alkylate specifically cysteine 67 of HLA-B27. Neither free cysteine in solution nor an exposed cysteine on a class II MHC molecule can be alkylated, showing that specific recognition between the anchor side-chain pocket of an MHC class I protein and the designed ligand (propinquity) is necessary to induce the selective covalent reaction with the MHC class I molecule.

Affinity Labels↗

PubMed1996

Crystallization and preliminary X-ray diffraction studies of the influenza C virus glycoprotein.

Influenza C virus contains a single surface glycoprotein in its lipid envelope which is the hemagglutinin-esterase-fusion glycoprotein (HEF). HEF binds cell-surface receptors, is a receptor-destroying enzyme (a 9-O-acetylesterase), and mediates the fusion of virus and host cell membranes. A bromelain-released soluble form of HEF has been crystallized. Two different tetragonal forms have been identified from crystals with the same morphology [P(1(3))22, a = b = 154.5, c = 414.4 A, and P4(1(3))2(1)2, a = b = 217.4, c = 421.4 A]. Both crystal forms share a common packing scheme. Synchrotron data collection and flash cooling of crystals have been used for high-resolution data collection.

Journal Article↗

PubMed1996

Direct binding of a soluble natural killer cell inhibitory receptor to a soluble human leukocyte antigen-Cw4 class I major histocompatibility complex molecule.

Natural killer (NK) cells expressing specific p58 NK receptors are inhibited from lysing target cells that express human leukocyte antigen (HLA)-C class I major histocompatibility complex molecules. To investigate the interaction between p58 NK receptors and HLA-Cw4, the extracellular domain of the p58 NK receptor specific for HLA-Cw4 was overexpressed in Escherichia coli and refolded from purified inclusion bodies. The refolded NK receptor is a monomer in solution. It interacts specifically with HLA-Cw4, blocking the binding of a p58-Ig fusion protein to HLA-Cw4-expressing cells, but does not block the binding of a p58-Ig fusion protein specific for HLA-Cw3 to HLA-Cw3-expressing cells. The bacterially expressed extracellular domain of HLA-Cw4 heavy chain and beta2-microglobulin were refolded in the presence of a HLA-Cw4-specific peptide. Direct binding between the soluble p58 NK receptor and the soluble HLA-Cw4-peptide complex was observed by native gel electrophoresis. Titration binding assays show that soluble monomeric receptor forms a 1:1 complex with HLA-Cw4, independent of the presence of Zn2+. The formation of complexes between soluble, recombinant molecules indicates that HLA-Cw4 is sufficient for specific ligation by the NK receptor and that neither glycoprotein requires carbohydrate for the interaction.

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

Antigenic peptides containing large PEG loops designed to extend out of the HLA-A2 binding site form stable complexes with class I major histocompatibility complex molecules.

Recognition of peptides bound to class I major histocompatibility complex (MHC) molecules by specific receptors on T cells regulates the development and activity of the cellular immune system. We have designed and synthesized de novo cyclic peptides that incorporate PEG in the ring structure for binding to class I MHC molecules. The large PEG loops are positioned to extend out of the peptide binding site, thus creating steric effects aimed at preventing the recognition of class I MHC complexes by T-cell receptors. Peptides were synthesized and cyclized on polymer support using high molecular weight symmetrical PEG dicarboxylic acids to link the side chains of lysine residues substituted at positions 4 and 8 in the sequence of the HLA-A2-restricted human T-lymphotrophic virus type I Tax peptide. Cyclic peptides promoted the in vitro folding and assembly of HLA-A2 complexes. Thermal denaturation studies using circular dichroism spectroscopy showed that these complexes are as stable as complexes formed with antigenic peptides.

Amino Acid Sequence↗

PubMed1996

The ectodomain of HIV-1 env subunit gp41 forms a soluble, alpha-helical, rod-like oligomer in the absence of gp120 and the N-terminal fusion peptide.

The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein is composed of a soluble glycopolypeptide gp120 and a transmembrane glycopolypeptide gp41. These subunits form non-covalently linked oligomers on the surface of infected cells, virions and cells transfected with the complete env gene. Two length variants of the extracellular domain of gp41 (aa 21-166 and aa 39-166), that both lack the N-terminal fusion peptide and the C-terminal membrane anchor and cytoplasmic domain, have been expressed in insect cells to yield soluble oligomeric gp41 proteins. Oligomerization was confirmed by chemical cross-linking and gel filtration. Electron microscopy and circular dichroism measurements indicate a rod-like molecule with a high alpha-helical content and a high melting temperature (78 degrees C). The binding of monoclonal antibody Fab fragments dramatically increased the solubility of both gp41 constructs. We propose that gp41 folds into its membrane fusion-active conformation, when expressed alone.

Animals↗

PubMed1996

A surface plasmon resonance assay for the binding of influenza virus hemagglutinin to its sialic acid receptor.

We have developed a sensitive microscale binding assay to study the interaction between influenza hemagglutinin and its cell surface receptor sialic acid using real-time surface plasmon resonance. The glycoprotein fetuin was bound to a carboxymethylated-Dextran sensor surface using N-hydroxysuccinimide and N-ethyl-N'-(dimethylaminopropyl) carbodiimide. Low-pH-induced BHA rosettes bind specifically to the fetuin-derivitized sensor surface, but not to an asialofetuin-derivitized sensor surface. Binding can be inhibited by preincubation of BHA rosettes with millimolar concentrations of inhibitors of the influenza hemagglutinin-sialic acid interaction. The association rate, dissociation rate, and dissociation constant for the multivalent interaction between BHA rosettes and the fetuin-derivitized sensor surface were also measured, allowing us to quantitate the tight binding achieved through the multivalent interaction between BHA rosettes and the fetuin-derivitized sensor surface.

Hemagglutinins, Viral↗

PubMed1996

Crystallographic analysis of endogenous peptides associated with HLA-DR1 suggests a common, polyproline II-like conformation for bound peptides.

The structure of the human major histocompatibility complex (MHC) class II molecule HLA-DR1 derived from the human lymphoblastoid cell line LG-2 has been determined in a complex with the Staphylococcus aureus enterotoxin B superantigen. The HLA-DR1 molecule contains a mixture of endogenous peptides derived from cellular or serum proteins bound in the antigen-binding site, which copurify with the class II molecule. Continuous electron density for 13 amino acid residues is observed in the MHC peptide-binding site, suggesting that this is the core length of peptide that forms common interactions with the MHC molecule. Electron density is also observed for side chains of the endogenous peptides. The electron density corresponding to peptide side chains that interact with the DR1-binding site is more clearly defined than the electron density that extends out of the binding site. The regions of the endogenous peptides that interact with DRI are therefore either more restricted in conformation or sequence than the peptide side chains or amino acids that project out of the peptide-binding site. The hydrogen-bond interactions and conformation of a peptide model built into the electron density are similar to other HLA-DR-peptide structures. The bound peptides assume a regular conformation that is similar to a polyproline type II helix. The side-chain pockets and conserved asparagine residues of the DR1 molecule are well-positioned to interact with peptides in the polyproline type II conformation and may restrict the range of acceptable peptide conformations.

Amino Acid Sequence↗