Biomedical subjects

D C Wiley

Publications and source records attributed to D C Wiley.

At least 19 recordsLinked to original sources

PubMed1992

Different length peptides bind to HLA-Aw68 similarly at their ends but bulge out in the middle.

We report here the determination and refinement to 1.9 A resolution by X-ray cryo-crystallography the structure of HLA-Aw68. The averaged image from the collection of bound, endogenous peptides clearly shows the atomic structure at the first three and last two amino acids in the peptides but no connected electron density in between. This suggests that bound peptides, held at both ends, take alternative pathways and could be of different lengths by bulging out in the middle. Peptides eluted from HLA-Aw68 include peptides of 9, 10 and 11 amino acids, a direct indication of the length heterogeneity of tightly bound peptides. Peptide sequencing shows relatively conserved 'anchor' residues at position 2 and the carboxy-terminal residue. Conserved binding sites for the peptide N and C termini at the ends of the class I major histocompatibility complex binding groove are apparently dominant in producing the long half-lives of peptide binding and the peptide-dependent stabilization of the class I molecule's structure.

Amino Acid Sequence↗

PubMed1992

Atomic structure of a human MHC molecule presenting an influenza virus peptide.

Infection by influenza virus results in the stimulation of cytotoxic T lymphocytes specific for killing virally infected cells. Specificity is provided by clonally distributed, hypervariable T-cell receptors on cytotoxic T lymphocytes which react with peptide fragments that are derived from viral proteins expressed in the cytoplasm and 'presented' on the surface of infected cells, bound to class I histocompatibility glycoproteins. Here we describe the structure of the complex between the human class I histocompatibility glycoprotein HLA-Aw68 and the influenza virus nucleoprotein peptide Np 91-99 as determined by X-ray cryocrystallography. Residues at both ends of the peptide are substantially buried in the peptide binding-site, whereas those in the middle of the peptide, P4 to P8, are predominantly exposed and could be recognized directly by T-cell receptors. The extended conformation of the bound viral peptide is remarkably similar to that of a collection of endogenous peptides with a different sequence motif bound to another human allele, HLA-B27. The structure defines in atomic detail the antigenic surface constructed of major histocompatibility complex and viral peptide atoms that is recognized by T-cell receptors.

Amino Acid Sequence↗

PubMed1992

Binding of influenza virus hemagglutinin to analogs of its cell-surface receptor, sialic acid: analysis by proton nuclear magnetic resonance spectroscopy and X-ray crystallography.

The interaction between influenza virus hemagglutinin and its cell-surface receptor, 5-N-acetylneuraminic acid (sialic acid), was probed by the synthesis of 12 sialic acid analogs, including derivatives at the 2-carboxylate, 5-acetamido, 4-, 7-, and 9-hydroxyl, and glycosidic positions. The equilibrium dissociation constants of these analogs were determined by nuclear magnetic resonance spectroscopy. Ligand modifications that reduced or abolished binding included the replacement of the 2-carboxylate with a carboxamide, the substitution of azido or N-benzyloxycarbonyl groups for the 5-acetamido group, and the replacement of the 9-hydroxyl with amino or O-acetyl moieties. Modifications having little effect on binding included the introduction of longer chains at the 4-hydroxyl position, the replacement of the acetamido methyl group with an ethyl group, and the removal of the 7-hydroxyl group. X-ray diffraction studies yielded 3 A resolution crystal structures of hemagglutinin in complex with four of the synthetic analogs [alpha-2-O-methyl-, 4-O-acetyl-alpha-2-O-methyl-, 9-amino-9-deoxy-alpha-2-O-methyl-, and alpha-2-O-(4'-benzylamidocarboxybutyl)-N-acetylneuraminic acid] and with the naturally occurring cell-surface saccharide (alpha 2-3)sialyllactose. The X-ray studies unambiguously establish the position and orientation of bound sialic acid, indicate the position of the lactose group of (alpha 2-3)sialyllactose, and suggest the location of an alpha-glycosidic chain (4'-benzylamidocarboxybutyl) that increases the binding affinity of sialic acid by a factor of about 3. Although the protein complexed with alpha-2-O-methylsialic acid contains the mutation Gly-135-->Arg near the ligand binding site, the mutation apparently does not affect the ligand's position. The X-ray studies allow us to interpret the binding affinities in terms of the crystallographic structure. The results suggest further experiments which could lead to the design of tight binding inhibitors of possible therapeutic value.

Amino Acid Sequence↗

PubMed1992

The three-dimensional structure of HLA-B27 at 2.1 A resolution suggests a general mechanism for tight peptide binding to MHC.

Cell surface complexes of class I MHC molecules and bound peptide antigens serve as specific recognition elements controlling the cytotoxic immune response. The 2.1 A structure of the human class I MHC molecule HLA-B27 provides a detailed composite image of a co-crystallized collection of HLA-B27-bound peptides, indicating that they share a common main-chain structure and length. It also permits direct visualization of the conservation of arginine as an "anchor" side chain at the second peptide position, which is bound in a potentially HLA-B27-specific pocket and may therefore have a role in the association of HLA-B27 with several diseases. Tight peptide binding to class I MHC molecules appears to result from the extensive contacts found at the ends of the cleft between peptide main-chain atoms and conserved MHC side chains, which also involve the peptide in stabilizing the three-dimensional fold of HLA-B27. The concentration of binding interactions at the peptide termini permits extensive sequence (and probably some length) variability in the center of the peptide, where it is exposed for T cell recognition.

Amino Acid Sequence↗

PubMed1992

HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides.

The two subunits of the human class I histocompatibility antigen (HLA)-A2 have been expressed at high levels (20-30 mg/liter) as insoluble aggregates in bacterial cells. The aggregates were dissolved in 8 M urea and then refolded to form an HLA-A2-peptide complex by removal of urea in the presence of an antigenic peptide. Two peptides from the matrix protein and nucleoprotein of influenza virus are known to bind to HLA-A2, and both support the refolding of the recombinant HLA-A2 molecule. An additional peptide, a nonamer from the gp120 envelope protein of human immunodeficiency virus type 1, also supported refolding. Yields of purified recombinant HLA-A2 are 10-15%. In the absence of an HLA-A2-restricted peptide, a stable HLA-A2 complex was not formed. Monoclonal antibodies known to bind to native HLA-A2 also bound to the recombinant HLA-A2-peptide complex. Three purified HLA-A2-peptide complexes refolded from bacterially produced protein aggregates crystallize under the identical conditions as HLA-A2 purified from human lymphoblastoid cells. Crystals of the recombinant HLA-A2 molecule in complex with the influenza matrix nonamer peptide, Mp(58-66), diffract to greater than 1.5-A resolution.

Amino Acid Sequence↗

PubMed1992

Introduction of intersubunit disulfide bonds in the membrane-distal region of the influenza hemagglutinin abolishes membrane fusion activity.

Influenza virus hemagglutinin (HA) mediates viral entry into cells by a low pH-induced membrane fusion event in endosomes. A number of structural changes occur throughout the length of HA at the pH of fusion. To probe their significance and their necessity for fusion activity, we have prepared a site-directed mutant HA containing novel intersubunit disulfide bonds designed to cross-link covalently the membrane-distal domains of the trimer. These mutations inhibited the low pH-induced conformational changes and prevented HA-mediated membrane fusion; conditions that reduced the novel disulfide bonds restored membrane fusion activity. We conclude that structural rearrangements in the membrane distal region of the HA are required for membrane fusion activity.

Animals↗

PubMed1992

The human class II MHC protein HLA-DR1 assembles as empty alpha beta heterodimers in the absence of antigenic peptide.

We have produced the human class II histocompatibility protein, HLA-DR1, as a soluble, secreted glycoprotein in insect cells infected with baculoviruses carrying truncated alpha and beta subunit genes. The peptide-binding site is empty, and the empty molecules are fully competent to bind antigenic peptide. We used the empty molecules to measure an intrinsic rate for peptide association, and to investigate the role of peptide in stabilizing the class II structure. Peptide binding kinetics for the empty molecule are only 10-fold faster than for peptide exchange into an occupied site, suggesting that a conformational change may accompany peptide binding. The native alpha beta heterodimer assembles in the absence of antigenic peptide, but peptide binding stabilizes the empty heterodimer against aggregation and against SDS-induced denaturation.

Amino Acid Sequence↗

PubMed1992

Crystallographic detection of a second ligand binding site in influenza virus hemagglutinin.

X-ray crystal structures have been determined for several complexes between influenza virus hemagglutinin and derivatives of its cell-surface receptor, sialic acid (Neu5Ac). Difference electron density maps establish the existence of a second binding site in addition to the primary site characterized previously. Three compounds bind to both sites: Neu5Ac(alpha 2-3)Gal(beta 1-4)Glc [(alpha 2-3)sialyllactose], alpha-2-O-(4'-benzylamidocarboxybutyl)-5-N-acetylneuraminic acid, and alpha-2-O-(4'-methylamidocarboxybutyl)-5-N-acetylneuraminic acid; and four other compounds bind only to the primary site: Neu5Ac(alpha 2-6)Gal(beta 1-4)Glc [(alpha 2-6)sialyllactose], alpha-2-O-methyl-5-N-acetylneuraminic acid, 4-]-acetyl-alpha-2-O-methyl-5-N-acetylneuraminic acid, and 9-amino-9-deoxy-alpha-2-O-methyl-5-N-acetylneuraminic acid. The maps also extend earlier results by showing the location of all three sugar residues of (alpha 2-3)sialyllactose in the primary binding site. The affinity of (alpha 2-3)sialyllactose for the second site was estimated by collecting x-ray diffraction data at various ligand concentrations and was found to be at least four times weaker than its affinity for the primary site. Although it is not yet known whether the second binding site participates in the infection process, it nevertheless offers a potential target for the design of antiviral drugs.

Binding Sites↗

PubMed1992

Truncated variants of gp120 bind CD4 with high affinity and suggest a minimum CD4 binding region.

The envelope glycoprotein, gp120, of human immunodeficiency virus type 1 (HIV-1) binds the cellular protein CD4 with high affinity. By deletion we show that 62 N- and 20 C-terminal residues along with the V1, V2 and V3 variable regions of gp 120 are unnecessary for CD4 binding. A 287 residue variant (ENV59), missing those 197 amino acids, binds to CD4 with high affinity. A polyclonal antibody failed to efficiently precipitate ENV59 which is consistent with the loss of immunodominant antigenic structures in the regions deleted. This suggests that ENV59 may have potential as an immunogen, able to elicit antibodies against more conserved regions of gp120. Additionally, complementing co-expressed gp120 fragments as well as a circularly permuted molecule bind CD4, and suggest either that the molecular termini are adjacent in the folded structure, or that an N-terminal region folds into the structure unconstrained by its method of attachment to the rest of the molecule.

Animals↗

PubMed1992

Crystallization and preliminary X-ray diffraction studies of the human major histocompatibility antigen HLA-B27.

The class I major histocompatibility (MHC) antigen HLA-B27 was purified by immunoaffinity chromatography from the homozygous human B lymphoblastoid cell line LG-2. Detergent-soluble HLA-B27 was cleaved with the protease papain to remove the hydrophobic transmembrane region and the cytoplasmic tail. Crystals of the resulting water-soluble extracellular fragments were obtained in hanging drops by the vapor-diffusion method. The crystals are triclinic, space group P1, with unit cell dimensions a = 45.9 A, b = 71.0 A, c = 83.7 A, alpha = 79.4 degrees, beta = 88.5 degrees, gamma = 89.9 degrees, and diffract beyond 2.5 A resolution.

Cell Line, Transformed↗

PubMed1992

Proton nuclear magnetic resonance studies of the binding of sialosides to intact influenza virus.

The dissociation constants for binding of sialic acid derivatives to the hemagglutinin on intact influenza virus were determined using nuclear magnetic resonance (NMR) spectroscopy. The dissociation constants determined with whole virus are similar to, but slightly higher than, those determined with BHA (hemagglutinin released from virus by treatment with the protease bromelain; Sauter et al., 1989, Biochemistry 28, 8388-8396), indicating that the sialic acid binding site is not significantly altered when hemagglutinin is released from virus. Binding was quantified by observing the concentration-dependent broadening of the sialoside resonances in the presence of X-31 virus or alternatively by observing the effect of the sialoside on the resonances of a competitive "reporter" ligand. The glycosidic substituent attached to the sialic acid makes relatively little difference in the affinity of the sialoside for virus: alpha(2,6)-sialyllactose (KD = 2.7 mM) binds only slightly more tightly than alpha(2,3)-sialyllactose (KD = 3.5 mM). However, inversion of the glycosidic center produces a dramatic change in affinity: the dissociation constant for the alpha-methyl glycoside of sialic acid is 4.2 mM, but not binding is observed with the beta-methyl glycoside.

Binding, Competitive↗

PubMed1992

An HLA-A2/beta 2-microglobulin/peptide complex assembled from subunits expressed separately in Escherichia coli.

The human class I histocompatibility antigen HLA-A2 has been assembled from subunits expressed separately in E. coli. A peptide that is known to be recognized by human cytotoxic T lymphocytes (CTLs) in association with HLA-A2 is a necessary component of the reconstitution mixture. The N-terminal extracellular fragment of the HLA-A2 heavy chain is initially synthesised as an insoluble aggregate. The aggregate is solubilized in denaturant, mixed with the influenza nucleoprotein 85-94 decapeptide (NP peptide), and diluted into a solution containing human beta 2-microglobulin (beta 2 m) isolated from the E. coli periplasm. The HLA-A2 heavy chain becomes soluble in physiological solutions if both beta 2m and the NP peptide are present. The reconstituted HLA-A2 complex is recognised by a monoclonal antibody that is specific for the native HLA-A2/beta 2m heterodimer, and is also recognised by a monoclonal antibody that recognises beta 2m. When other peptides known from CTL studies to associate with HLA-A2 are used, a significantly lower yield of reconstituted complex is obtained. The isoelectric point of the reconstituted complex depends on which peptide is used, confirming that the peptide is a component of the reconstituted complex.

Amino Acid Sequence↗

PubMed1991

CD4-binding regions of human immunodeficiency virus envelope glycoprotein gp120 defined by proteolytic digestion.

The gp120 envelope glycoprotein of human immunodeficiency virus type 1 binds the cell surface protein CD4 with high affinity. Here we report the use of proteolysis to define regions of gp120 involved in CD4 binding. Cleavage of gp120 with Staphylococcus aureus V8 protease at residue 269 or with trypsin at residue 432 destroys CD4 binding. These same sites are protected from proteolytic cleavage by bound CD4. Cleavages at 64, 144, 166, 172, and 315 do not affect binding and are not protected by bound CD4, indicating that these regions are not critical for binding CD4. All proteolytic fragments found in coprecipitates with CD4 were covalently associated via disulfides and comprised complete gp120 molecules. Previous conclusions by Nygren et al. [Nygren, A., Bergman, T., Matthews, T., Jornvall, H. & Wigzell, H. (1988) Proc. Natl. Acad. Sci. USA 85, 6543-6546] that both large and small (95-kDa and 25-kDa) V8 proteolytic fragments bind CD4, independently, are not distinguished by their experiments from the result found here that the small fragment immunoprecipitates with CD4 while disulfide-linked to the larger fragment.

Amino Acid Sequence↗

PubMed1991

Amantadine selection of a mutant influenza virus containing an acid-stable hemagglutinin glycoprotein: evidence for virus-specific regulation of the pH of glycoprotein transport vesicles.

Mutants of influenza Rostock virus (H7N1 subtype) were selected for resistance to amantadine hydrochloride at concentrations of the antiviral drug known to affect the function of the virus M2 transmembrane protein. Sequence analysis revealed that three mutants had no changes in M2 but contained a lysine to isoleucine substitution in the hemagglutinin (HA) membrane glycoprotein at position 58 of HA2. The mutant viruses were found to fuse membranes at a pH value 0.7 lower than wild type and to exhibit changes in the conformation of their HAs specifically at the lower pH. The homologous lysine to isoleucine substitution was introduced by site-specific mutagenesis into the HA of X-31 influenza virus (H3 subtype), which was expressed by using vaccinia virus recombinants. The expressed HA also mediated membrane fusion and changed in conformation at a pH value 0.7 lower than wild type. These results indicate that increased acid stability of the HA obviates the consequences of the inhibition of M2 function by amantadine and provide further evidence for the role of M2 in regulating the pH of vesicles involved in glycoprotein transport to the cell surface.

Amantadine↗

PubMed1991

Ligand recognition by influenza virus. The binding of bivalent sialosides.

Infection by influenza virus is initiated by a cellular adhesion event that is mediated by the viral protein, hemagglutinin, which is exposed on the surface of the virion. Hemagglutinin recognizes and binds to cell surface sialic acid residues. Although each individual ligand binding interaction is weak, the high affinity of influenza virus for cells that bear sialic acid residues is thought to result from a multivalent attachment process involving many similar recognition events. To evaluate such binding we have synthesized three series of compounds, each containing two sialic acid residues separated by spacers of different length, and have tested them as ligands for influenza hemagglutinin. No increased binding to the bromelain-released hemagglutinin ectodomain was seen for any of the bivalent compounds as determined by 1H NMR titration. In contrast, however, a spacer length between sialic acid residues of approximately 55 A sharply increases the binding of these bidentate species to whole virus as determined by hemagglutination inhibition assays. The most effective compound containing glycines in the linking chain displayed 100-fold increased affinity for whole virus over the paradigm monovalent ligand, Neu5Ac alpha 2Me.

Animals↗

PubMed1991

The structure of HLA-B27 reveals nonamer self-peptides bound in an extended conformation.

X-ray crystallography reveals electron density in the antigen-binding site of HLA-B27 that is an interpretable image of nonameric peptides in a largely extended conformation. Clear density exists for the main chain and several side chains and is consistent with the sequence of 11 nonameric self-peptides eluted from HLA-B27. Pockets in the antigen-binding cleft bind four side chains and the amino and carboxyl termini of the peptide.

Amino Acid Sequence↗

PubMed1991

Identification of self peptides bound to purified HLA-B27.

A pool of endogenous peptides bound to the human class I MHC molecule, HLA-B27, has been isolated. Microsequence analysis of the pool and of 11 HPLC-purified peptides provides information on the binding specificity of the HLA-B27 molecule. The peptides all seem to be nonamers, seven of which match to protein sequences in a database search. These self peptides derive from abundant cytosolic or nuclear proteins, such as histone, ribosomal proteins, and members of the 90K heat-shock protein family.

Amino Acid Sequence↗

PubMed1991

Refined structure of the human histocompatibility antigen HLA-A2 at 2.6 A resolution.

The three-dimensional structure of the human histocompatibility antigen HLA-A2 was determined at 3.5 A resolution by a combination of isomorphous replacement and iterative real-space averaging of two crystal forms. The monoclinic crystal form has now been refined by least-squares methods to an R-factor of 0.169 for data from 6 to 2.6 A resolution. A superposition of the structurally similar domains found in the heterodimer, alpha 1 onto alpha 2 and alpha 3 onto beta 2m, as well as the latter pair onto the ancestrally related immunoglobulin constant domain, reveals that differences are mainly in the turn regions. Structural features of the alpha 1 and alpha 2 domains, such as conserved salt-bridges that contribute to stability, specific loops that form contacts with other domains, and the antigen-binding groove formed from two adjacent helical regions on top of an eight-stranded beta-sheet, are analyzed. The interfaces between the domains, especially those between beta 2m and the HLA heavy chain presumably involved in beta 2m exchange and heterodimer assembly, are described in detail. A detailed examination of the binding groove confirms that the solvent-accessible amino acid side-chains that are most polymorphic in mouse and human alleles fill up the central and widest portion of the binding groove, while conserved side-chains are clustered at the narrower ends of the groove. Six pockets or sub-sites in the antigen-binding groove, of diverse shape and composition, appear suited for binding side-chains from antigenic peptides. Three pockets contain predominantly non-polar atoms; but others, especially those at the extreme ends of the groove, have clusters of polar atoms in close proximity to the "extra" electron density in the binding site. A possible role for beta 2m in stabilizing permissible peptide complexes during folding and assembly is presented.

Amino Acid Sequence↗