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D C Wiley

Publications and source records attributed to D C Wiley.

At least 55 records · Page 3Linked to original sources
PubMed1999

X-ray crystallographic determination of the structure of the influenza C virus haemagglutinin-esterase-fusion glycoprotein.

The structure of the haemagglutinin-esterase-fusion (HEF) glycoprotein from influenza C virus has been determined to 3.2 A resolution by X-ray crystallography. A synthetic mercury-containing esterase inhibitor and receptor analogue, 9-acetamidosialic acid alpha-thiomethylmercuryglycoside, was designed as the single isomorphous heavy-atom derivative. The asymmetric unit of one crystal form (form I; P4322, a = b = 155.4, c = 414.4 A) contained an HEF trimer. Six mercury sites identifying the three haemagglutination and three esterase sites were located by difference Patterson map analysis of a 6.5 A resolution derivative data set. These positions defined the molecular threefold-symmetry axis of the HEF trimer. A molecular envelope was defined by averaging a 7.0 A resolution electron-density map, phased by single isomorphous replacement (SIR), about the non-crystallographic threefold-symmetry axis. Iterative non-crystallographic symmetry averaging in real space, solvent flattening and histogram matching were used to extend the phases to 3.5 A resolution. Molecular replacement of the model into a second crystal form (form II; P43212, a = b = 217.4, c = 421.4 A) containing two HEF trimers per asymmetric unit permitted iterative ninefold averaging of the electron density. The 3.5 A electron-density map allowed an unambiguous tracing of the polypeptide chain and identification of N-linked carbohydrates. The model has been refined by least squares to 3.2 A resolution (Rfree = 26.7%).

Acetylesterase↗
PubMed1999

Why adolescent boys dream of becoming professional athletes.

A review of studies which investigated drawings of the ideal man and the occupational aspirations of boys (ages 11-18 years) from several countries indicated that becoming a professional athlete was a popular but unrealistic aspiration for many adolescent boys. Boys who were athletes and nonathletes from diverse ethnic groups and nationalities dreamed of becoming professional athletes. In two additional studies in the United States of America, adolescents were asked why they thought boys most often selected professional athlete as a possible future occupation. Adolescents perceived professional athletes as rich, famous, and glorified. Enhancement of status and financial gain were ranked as more important than the desire to play sports.

Adolescent↗
PubMed1998

Structure of a trimeric domain of the MHC class II-associated chaperonin and targeting protein Ii.

The invariant chain (Ii) plays a critical role in MHC class II antigen processing by stabilizing peptide-free class II alphabeta heterodimers in a nonameric (alphabetaIi)3 complex soon after their synthesis and directing transport of the complex from the endoplasmic reticulum to compartments where peptide loading of class II takes place. Loading progresses following Ii proteolysis and via an intermediate complex of MHC class II with an Ii-derived peptide, CLIP. CLIP is substituted by exogenous peptidic fragments in an exchange reaction catalyzed by HLA-DM. The CLIP region of Ii, roughly residues 81-104, is one of two segments shown to interact with class II HLA-DR molecules. The other segment, Ii 118-216, is C-terminal to CLIP, mediates trimerization of the ectodomain of Ii and interferes with DM/class II binding. Here we report the three-dimensional structure of this trimeric domain, determined by nuclear magnetic resonance (NMR) studies of a 27 kDa trimer of human Ii 118-192. The cylindrical shape of the molecule and the mapping of conserved residues delimit surfaces which may be important for interactions between Ii and class II molecules.

Amino Acid Sequence↗
PubMed1998

Structure of the haemagglutinin-esterase-fusion glycoprotein of influenza C virus.

The spike glycoproteins of the lipid-enveloped orthomyxoviruses and paramyxoviruses have three functions: to recognize the receptor on the cell surface, to mediate viral fusion with the cell membrane, and to destroy the receptor. In influenza C virus, a single glycoprotein, the haemagglutinin-esterase-fusion (HEF) protein, possesses all three functions. In influenza A and B, the first two activities are mediated by haemagglutinin and the third by a second glycoprotein, neuraminidase. Here we report the crystal structure of the HEF envelope glycoprotein of influenza C virus. We have identified the receptor-binding site and the receptor-destroying enzyme (9-O-acetylesterase) sites, by using receptor analogues. The receptor-binding domain is structurally similar to the sialic acid-binding domain of influenza A haemagglutinin, but binds 9-O-acetylsialic acid. The esterase domain has a structure similar to the esterase from Streptomyces scabies and a brain acetylhydrolase. The receptor domain is inserted into a surface loop of the esterase domain and the esterase domain is inserted into a surface loop of the stem. The stem domain is similar to that of influenza A haemagglutinin, except that the triple-stranded, alpha-helical bundle diverges at both of its ends, and the amino terminus of HEF2, the fusion peptide, is partially exposed. The segregation of HEF's three functions into structurally distinct domains suggests that the entire stem region, including sequences at the amino and carboxy termini of HEF1 which precede the post-translational cleavage site between HEF1 and HEF2, forms an independent fusion domain which is probably derived from an ancestral membrane fusion protein.

Acetylesterase↗
PubMed1998

Structure of the hemagglutinin precursor cleavage site, a determinant of influenza pathogenicity and the origin of the labile conformation.

The membrane fusion potential of influenza HA, like many viral membrane-fusion glycoproteins, is generated by proteolytic cleavage of a biosynthetic precursor. The three-dimensional structure of ectodomain of the precursor HA0 has been determined and compared with that of cleaved HA. The cleavage site is a prominent surface loop adjacent to a novel cavity; cleavage results in structural rearrangements in which the nonpolar amino acids near the new amino terminus bury ionizable residues in the cavity that are implicated in the low-pH-induced conformational change. Amino acid insertions at the cleavage site in HAs of virulent avian viruses and those of viruses isolated from the recent severe outbreak of influenza in humans in Hong Kong would extend this surface loop, facilitating intracellular cleavage.

Bromelains↗
PubMed1998

Crystal structure of HLA-DR2 (DRA*0101, DRB1*1501) complexed with a peptide from human myelin basic protein.

Susceptibility to multiple sclerosis is associated with the human histocompatibility leukocyte antigen (HLA)-DR2 (DRB1*1501) haplotype. The structure of HLA-DR2 was determined with a bound peptide from human myelin basic protein (MBP) that is immunodominant for human MBP-specific T cells. Residues of MBP peptide that are important for T cell receptor recognition are prominent, solvent exposed residues in the crystal structure. A distinguishing feature of the HLA-DR2 peptide binding site is a large, primarily hydrophobic P4 pocket that accommodates a phenylalanine of the MBP peptide. The necessary space for this aromatic side chain is created by an alanine at the polymorphic DRbeta 71 position. These features make the P4 pocket of HLA-DR2 distinct from DR molecules associated with other autoimmune diseases.

Binding Sites↗
PubMed1998

Crystal structures of HLA-A*0201 complexed with antigenic peptides with either the amino- or carboxyl-terminal group substituted by a methyl group.

The crystal structures of class I major histocompatibility complex (MHC) molecules complexed with antigenic peptides revealed a network of hydrogen bonds between the charged amino- and carboxyl-termini of the peptides and conserved MHC residues at both ends of the peptide binding site. These interactions were shown to contribute substantially to the stability of class I MHC/peptide complexes by thermal denaturation studies using synthetic peptides in which either the amino- or carboxyl-terminal group is substituted by a methyl group. Here we report crystal structures of HLA-A*0201 complexed with these terminally modified synthetic peptides showing that they adopt the same bound conformation as antigenic peptides. A number of variations in peptide conformation were observed for the terminally modified peptides, including in one case, a large conformational difference in four central peptide residues that is apparently caused by the lattice contact. This is reminiscent of the way binding a T-cell receptor changed the conformation of central residues of an MHC-bound peptide. The structures determined identify which conserved hydrogen bonds are eliminated in terminally substituted peptides and suggest an increased energetic importance of the interactions at the peptide termini for MHC-peptide stability.

Crystallography, X-Ray↗
PubMed1998

A polar octapeptide fused to the N-terminal fusion peptide solubilizes the influenza virus HA2 subunit ectodomain.

As a step toward studying membrane fusion with a simplified molecule, the ectodomain, residues 1-185, of the membrane-anchored subunit HA2 of the influenza virus haemagglutinin (HA) was solubilized by adding the very polar FLAG octapeptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) to the N-terminal HA2 fusion peptide. The resulting chimeric protein, F185, when expressed in bacteria, folded spontaneously into a soluble trimer, with a high alpha-helical content and a high melting temperature, structural characteristics of the low-pH-induced conformation of HA2. Removal of the FLAG octapeptide by proteolysis with enterokinase converted the soluble molecule to one that aggregated, bound nonionic detergent, and bound to lipid vesicles, properties of the low-pH-induced conformation of HA. Thermolysin treatment of the aggregated protein removed the nonpolar fusion peptide, regenerating soluble trimers of HA2 (residues 24-185), which is analogous to thermolysin treatment of HA in the low-pH-induced conformation. Thermolysin treatment also dissociates F185 from the detergent-protein complex by removing the fusion peptide. These results suggest that highly polar peptides can be fused to the membrane-binding regions of membrane proteins to increase their solubility. They also indicate that ectodomains of HA2 made in bacteria have membrane-binding properties similar to those of the same ectodomain generated by low-pH treatment of HA isolated from virus.

Amino Acid Sequence↗
PubMed1998

Expression and crystallization of the complex of HLA-DR2 (DRA, DRB1*1501) and an immunodominant peptide of human myelin basic protein.

HLA-DR2 is associated with susceptibility to multiple sclerosis (MS). A peptide from human myelin basic protein (MBP, residues 85-99) was previously found to bind to purified HLA-DR2 (DRA, DRB1*1501) and to be recognized by human MBP-specific T cell clones. Soluble HLA-DR2 was expressed in the baculovirus system by replacing the hydrophobic transmembrane regions and cytoplasmic segments of DRalpha and DRbeta with leucine zipper dimerization domains from the transcription factors Fos and Jun. In the expression construct, the MBP(85-99) sequence was covalently linked to the N terminus of the mature DRbeta chain. The recombinant protein was secreted by Sf9 cells infected with the recombinant baculovirus and purified by affinity chromatography. The leucine zipper dimerization domains were then cleaved from the assembled HLA-DR2/MBP peptide complex with V8 protease, and the protein was further purified by anion-exchange HPLC. Analysis by HPLC gel filtration indicated that the HLA-DR2/MBP peptide complex did not have a tendency to aggregate. The purified HLA-DR2/MBP peptide complex readily crystallized by the hanging drop method in 15-18% polyethylene glycol 6000/100 mM glycine, pH 3.5. At a synchrotron radiation source, a crystal with a tetragonal space group diffracted to a resolution of 2.6 A. The expression of such homogenous HLA-DR/peptide complexes may facilitate cocrystallization with T cell receptors as well as other molecules involved in T cell receptor recognition and signaling.

Amino Acid Sequence↗
PubMed1998

Zinc-dependent dimers observed in crystals of human endostatin.

The crystal structure of human endostatin reveals a zinc-binding site. Atomic absorption spectroscopy indicates that zinc is a constituent of both human and murine endostatin in solution. The human endostatin zinc site is formed by three histidines at the N terminus, residues 1, 3, and, 11, and an aspartic acid at residue 76. The N-terminal loop ordered around the zinc makes a dimeric contact in human endostatin crystals. The location of the zinc site at the amino terminus, immediately adjacent to the precursor cleavage site, suggests the possibility that the zinc may be involved in activation of the antiangiogenic activity following cleavage from the inactive collagen XVIII precursor or in the cleavage process itself.

Base Sequence↗
PubMed1998

The central structural feature of the membrane fusion protein subunit from the Ebola virus glycoprotein is a long triple-stranded coiled coil.

The ectodomain of the Ebola virus Gp2 glycoprotein was solubilized with a trimeric, isoleucine zipper derived from GCN4 (pIIGCN4) in place of the hydrophobic fusion peptide at the N terminus. This chimeric molecule forms a trimeric, highly alpha-helical, and very thermostable molecule, as determined by chemical crosslinking and circular dichroism. Electron microscopy indicates that Gp2 folds into a rod-like structure like influenza HA2 and HIV-1 gp41, providing further evidence that viral fusion proteins from diverse families such as Orthomyxoviridae (Influenza), Retroviridae (HIV-1), and Filoviridae (Ebola) share common structural features, and suggesting a common membrane fusion mechanism.

Amino Acid Sequence↗
PubMed1998

Studies of the binding properties of influenza hemagglutinin receptor-site mutants.

Site-specific mutations have been made in the influenza hemagglutinin (HA) receptor binding site to assess the contribution of individual amino acid residues to receptor recognition. Screening of mutant HAs, expressed using recombinant vaccinia virus-infected cells, for their abilities to bind human erythrocytes indicated that substitutions involving conserved residues Y98F, H183F, and L194A severely restricted binding and that the substitution W153A prevented cell surface expression of HA. Mutation of residues E190 and S228 that are in positions to form hydrogen bonds with the 9-OH of sialic acid appeared to increase erythrocyte binding slightly, as did the substitution G225R. Substitutions of other residues that are directly or indirectly involved in receptor binding, S136T, S136A, Y195F, G225D, and L226P, had intermediate effects on binding between these two extremes. Estimates of changes in receptor binding specificity based on inhibition of binding to erythrocytes by nonimmune horse sera indicated that mutants G225R and L226P, unlike wild-type HA, were not inhibited; Y195F and G225D mutants were, like wild type, inhibited; and erythrocyte binding by mutants S136A, S136T, E190A, and S228G was only partially inhibited. Viruses containing mutant HAs Y98F, S136T, G225D, and S228G that cover the range of erythrocyte binding properties observed were also constructed by transfection. All four transfectant viruses replicated in MDCK cells and embryonated hens' eggs as efficiently as wild-type X-31 virus, although the Y98F mutant virus was unable to agglutinate erythrocytes. Mutant MDCK cells that have reduced levels of cell surface sialic acids were susceptible to infection by S136T, G225D, and S228G transfectant viruses and by wild type but not by the Y98F transfectant virus.

Animals↗
PubMed1998

Two human T cell receptors bind in a similar diagonal mode to the HLA-A2/Tax peptide complex using different TCR amino acids.

The three-dimensional structure of a human alphabeta T cell receptor (TCR), B7, bound to the HLA-A2 molecule/HTLV-1 Tax peptide complex was determined by x-ray crystallography. Although different from the A6 TCR, previously studied, in 16 of the 17 residues that contact HLA-A2/Tax, the B7 TCR binds in a similar diagonal manner, only slightly tipped and rotated, relative to the A6 TCR. The structure explains data from functional assays on the specificity differences between the B7 and A6 TCRs for agonist, partial agonist, and null peptides. The existence of a structurally similar diagonal binding mode for TCRs favors mechanisms based on the formation of geometrically defined supramolecular assemblies for initiating signaling.

Amino Acid Sequence↗
PubMed1998

The structure of HLA-DM, the peptide exchange catalyst that loads antigen onto class II MHC molecules during antigen presentation.

The three-dimensional structure of the soluble ecto-domain of HLA-DM has been determined to 2.5 A resolution by X-ray crystallography. HLA-DM has both peptide exchange activity and acts as a chaperone to peptide-free class II MHC molecules. As predicted, the structure is similar to that of classical class II MHC molecules except that the peptide-binding site is altered to an almost fully closed groove. An unusual cavity is found at the center of the region that binds peptides in class II MHC molecules, and a tryptophanrich lateral surface is identified that is a candidate both for binding to HLA-DR, to effect catalysis, and to HLA-DO, an inhibitor.

Antigen Presentation↗
PubMed1998

Crystal structure of the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain.

We have determined the structure of GP2 from the Ebola virus membrane fusion glycoprotein by X-ray crystallography. The molecule contains a central triple-stranded coiled coil followed by a disulfide-bonded loop homologous to an immunosuppressive sequence in retroviral glycoproteins, which reverses the chain direction and connects to an alpha helix packed antiparallel to the core helices. The structure suggests that fusion peptides near the N termini form disulfide-bonded loops at one end of the molecule and that the C-terminal membrane anchors are at the same end. In this conformation, GP2 could both bridge two membranes and facilitate their apposition to initiate membrane fusion. We also find a heptad irregularity like that in low-pH-induced influenza HA2 and a solvent ion trapped in a coiled coil like that in retroviral TMs.

Amino Acid Sequence↗
PubMed1998

Structural characterization of a soluble and partially folded class I major histocompatibility heavy chain/beta 2m heterodimer.

Class I major histocompatibility (MHC) heavy chain (HC) must fold and assemble with beta 2 microglobulin (beta 2m) prior to binding peptides in the endoplasmic reticulum (ER). Each of these events is mediated by association with chaperones and other proteins and is an essential requirement for the maturation and normal cell surface expression of stable class I MHC-peptide complexes. Here we describe the biochemical and structural characterization of a soluble HC (B*0702)/beta 2m heterodimer, apparently free of peptide. Results suggest that the peptide binding domains (alpha 1 and alpha 2) of this folding intermediate are unstable and possess many of the properties ascribed to the molten globule state. The partially folded state of the HC/beta 2m heterodimer is consistent with the suggestion that it is stabilized by chaperones and other proteins in the ER. This soluble intermediate may be useful for studying protein-assisted folding and peptide binding of class I MHC molecules.

Circular Dichroism↗
PubMed1998

Using picture identification for research with preschool children.

Assessing preschool childrens' health knowledge and skill levels, and their recognition of health behavior, prove essential in developing and maintaining effective health education programs. However, the intent of the assessment process, and the methodology, must be carefully considered. Picture identification can be appropriately used to determine the status of a young child's health knowledge or skills and thus decide how the child might benefit from further learning experiences. Knowledge or skill assessment for young children primarily involves observing children while they demonstrate knowledge through an activity or behavior, although this assessment may be supplemented with a checklist or instrument, such as a picture identification instrument. This article 1) reviews the reliability of picture identification tools in previous studies; 2) provides recommendations for assessing health knowledge of preschool children; and 3) suggests ways health knowledge data can be used appropriately in early childhood education programs. The result of quality knowledge and skill assessment provides direction for program planners in developing appropriate health education programs for preschoolers.

Child, Preschool↗