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 α -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.
Class I major histocompatibility complex (MHC) molecules present peptides to CD8+ T cells for immunological surveillance (reviewed in ref. 1). The structures of complexes of class I MHC molecules ...with octamer, nonamer and decamer peptides determined until now show a common binding mode, with both peptide termini bound in conserved pockets at the ends of the peptide binding site. Length variations were accommodated by the peptide bulging or zig-zagging in the middle. Here we describe the structure of a decamer peptide which binds with the carboxy-terminal residue positioned outside the peptide binding site. Several protein side chains have rearranged to allow the peptide to exit. The structure suggests that even longer peptides could bind. The energetic effect of the altered mode of binding has been assessed by measuring the stability of the complex to thermal denaturation. Peptides bound in this novel manner are stable at physiological temperature, raising questions about their role in T-cell recognition and their production by proteolytic processing.
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.
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 Å 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 α(2→6) or α(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 NeuAcα(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 NeuAcα(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 α(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 NeuAcα(2→3)Galβ(1→4)Glc, NeuAcα(2→6)Galβ(1→4)Glc, NeuAcα(2→3)Galβ(1→3)GlcNAcβ(1→3)Galβ(1→4)Glc, NeuAcα(2→6)Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)Glc, and NeuAcα(2→6)Galβ(1→4)GlcNAc2β(1→3/6)Gal-β-O-(CH2)5-COOCH3.
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.
The invariant chain (Ii) plays a critical role in MHC class II antigen processing by stabilizing peptide‐free class II αβ heterodimers in a nonameric (αβIi)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.
Class II and class I histocompatibility molecules allow T cells to recognize 'processed' polypeptide antigens. The two polypeptide chains of class II molecules, alpha and beta, are each composed of ...two domains (for review see ref. 6); the N-terminal domains of each, alpha 1 and beta 1, are highly polymorphic and appear responsible for binding peptides at what appears to be a single site and for being recognized by MHC-restricted antigen-specific T cells. Recently, the three-dimensional structure of the foreign antigen binding site of a class I histocompatibility antigen has been described. Because a crystal structure of a class II molecule is not available, we have sought evidence in class II molecules for the structural features observed in the class I binding site by comparing the patterns of conserved and polymorphic residues of twenty-six class I and fifty-four class II amino acid sequences. The hypothetical class II foreign-antigen binding site we present is consistent with mutation experiments and provides a structural framework for proposing peptide binding models to help understand recent peptide binding data.
The three-dimensional structure of a Staphylococcus aureus superantigen, toxic shock syndrome toxin-1 (TSST-1), complexed with a human class II major histocompatibility molecule (DR1), was determined ...by x-ray crystallography. The TSST-1 binding site on DR1 overlaps that of the superantigen S. aureus enterotoxin B (SEB), but the two binding modes differ. Whereas SEB binds primarily off one edge of the peptide binding site of DR1, TSST-1 extends over almost one-half of the binding site and contacts both the flanking α helices of the histocompatibility antigen and the bound peptide. This difference suggests that the T cell receptor (TCR) would bind to TSST-1:DR1 very differently than to DR1:peptide or SEB:DR1. It also suggests that TSST-1 binding may be dependent on the peptide, though less so than TCR binding, providing a possible explanation for the inability of TSST-1 to competitively block SEB binding to all DR1 molecules on cells (even though the binding sites of TSST-1 and SEB on DR1 overlap almost completely) and suggesting the possibility that T cell activation by superantigen could be directed by peptide antigen.
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.
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