Structure of the concanavalin A-methyl α-D-mannopyranoside complex at 6-Å resolution

Hardman, Karl D.; Ainsworth, Clinton F.

doi:10.1021/bi00650a026

Cited by 129 publications

(43 citation statements)

References 28 publications

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“…These studies suggested that peptides might be found that competitively inhibit other classes of carbohydrate-binding proteins An amino-terminal library of random octapeptides consisting of =1.4 x 109 members (-5% of all possible octamers) was affinity-purified against immobilized Con A. When phage were eluted by a low-pH citrate buffer to dissociate the phage-Con A complex, the sequence Tyr-Xaa-Tyr (where Xaa is predominantly a hydrophilic amino acid) was Although a number of hydrophobic binding sites exist on the Con A surface, including sites for fluorescent dansyl derivatives (35,36), tryptophan or indoleacetic acid (37), and benzoic acid derivatives (38), a number of studies indicate that the chromogenic ligand p-nitrophenyl a-D-mannopyranoside binds at the saccharide binding site (18,39,40 (1, 42). The sugar interacts with the lectin through a network of seven hydrogen bonds that connect four oxygens of the ligand with five amino acid residues of the protein.…”

Section: Resultsmentioning

confidence: 99%

Peptide ligands for a sugar-binding protein isolated from a random peptide library.

Oldenburg

Loganathan

Goldstein

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

250

119

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Peptide Carbohydrate-protein interactions form the basis of a host of biological processes (1, 2). For example, the periplasmic monosaccharide-binding proteins of Gram-negative bacteria serve as receptors for transport and chemotaxis (3), while cell-surface lectins mediate such processes as cell-cell adhesion (4, 5) and lymphocyte migration through lymphoid tissues (6). Carbohydrate recognition is central to the enzymatic synthesis and degradation of polysaccharides, glycoproteins, and glycolipids that play essential roles in metabolism and in the maintenance of cellular structures. Carbohydrate-protein associations are also critical in certain cycles of viral infection. For example, binding of the hemagglutinin protein of human influenza virus to sialic acid residues on erythrocyte cell-surface glycoproteins represents the initial step in influenza infection (7).Consequently, the development of potent inhibitors of carbohydrate-specific proteins may be of considerable importance in the generation of new therapeutic agents. However, the synthesis of complex carbohydrate ligands and analogs often requires many time-consuming, low-yielding steps. Chemical synthesis of oligosaccharides requires sophisticated strategies for protecting/deprotecting and assembling sugar monomers and for controlling product regiochemistry and stereochemistry (8-10). Enzymatic synthesis using glycosyltransferases has emerged as a useful alternative to chemical synthesis (11,12). However, this approach is limited by the availability of enzymes with the appropriate specificities.An alternative approach to the synthesis of polysaccharide ligands for carbohydrate-specific receptors is to ask whether peptides can be found that bind these proteins with high affinities. We describe a strategy for identifying novel peptide ligands for carbohydrate-binding proteins based on the screening of a large, highly diverse peptide library expressed on the surface of filamentous phage fd (13-16). The library consists of phage bearing random octapeptides fused to the amino terminus of the minor coat protein, pIII, and was screened by affinity purification on immobilized receptor. We chose as a model system the lectin Con A fromjack bean, whose physicochemical properties have been extensively studied (17). Con A is a tetramer composed of four identical polypeptide chains consisting of 237 residues each. Con A, which interacts preferentially with oligosaccharides bearing terminal a-linked mannose or glucose residues, is frequently employed in the purification and structural characterization of carbohydrates and glycoproteins and is also a lymphocyte mitogen. We report the isolation of peptide ligands for Con A that prevent binding of known monosaccharide ligands of the lectin and that inhibit Con A-dependent precipitation of polysaccharides. The prospect that specific ligands for carbohydrate-binding proteins can be assembled by coupling amino acid building blocks (rather than sugars) should greatly reduce the synthetic difficulties associated with i...

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Section: Resultsmentioning

confidence: 99%

Peptide ligands for a sugar-binding protein isolated from a random peptide library.

Oldenburg

Loganathan

Goldstein

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

250

119

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“…The MS11 synthetic peptide analogues used in this study (Table 1) correspond to regions both of conserved sequence (residues [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35], 41-50, 48-60, and 69-84) and of variable sequence (residues 107-121, 121-134, and 135-151) between pilin from various gonococcal strains (12). Fourteen rabbits were immunized with the seven peptides conjugated to thyroglobulin (two rabbits per conjugate).…”

Section: Resultsmentioning

confidence: 99%

“…A high percentage of the residues that hydrogen-bond the ligand in the binding sites of the few carbohydrate-binding proteins whose three-dimensional structures have been determined (4,(24)(25)(26)(27) are present in turns. We felt that an antibody generated against a predicted turn in the conserved region of gonococcal pilin might block pili from binding eukaryotic membranes.…”

Section: Discussionmentioning

confidence: 99%

Antibodies to peptides corresponding to a conserved sequence of gonococcal pilins block bacterial adhesion.

Rothbard¹,

Fernandez²,

Wang³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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Antisera generated against each of seven synthetic peptides corresponding to constant and variable sequences of the pilin from gonococcal strain MS11 were assayed for their ability to crossreact with intact pili from both homologous and heterologous strains. The peptides elicited roughly equal antipeptide responses but varied substantially in their ability to elicit antisera that crossreacted with intact pili. Of the antisera to peptides corresponding to regions of conserved sequence, antisera directed against residues 69-84 were the most efficient in binding pili from all strains tested in both solid-phase assays and immunoblots. Anti-69-84 also efficiently precipitated a tryptic fragment of pilin known to bind human endocervical cells. Sera against the two peptides (121-134 and 135-151) previously shown to contain strain-specific epitopes crossreacted with MS11 pili equally well, but differed in their ability to bind pili from heterologous strains. Anti-121-134 was strain-specific whereas anti-135-151 bound all pilin tested. Each of the sera was examined for its ability to inhibit bacterial adhesion to a human endometrial carcinoma cell line. Sera generated against residues 41-50 and 69-84 successfully inhibited a heterologous gonococcal strain from binding. These peptides could be important components of an effective vaccine for the prevention of gonorrhea.The antigenic variation of the surface molecules of pathogenic organisms is an important obstacle to the development of efficacious vaccines for a variety of diseases (1-6). In only a few systems are the molecular details of the sequence variation of the membrane proteins and the genetic mechanism for their variation partially understood (7,8).Efforts to develop a successful vaccine against gonorrhea have concentrated on blocking the organism's binding to eukaryotic cell surfaces. Gonococcal adhesion to mucus membranes is mediated in part by pili, filamentous polymers that extend out from the bacterial surface (9). These organelles are composed of a single repeating protein subunit, pilin. Pili prepared from different gonococcal strains are antigenically heterogeneous and attempts to use them as components of a vaccine have resulted only in protection against the homologous strain (10). In an earlier investigation, we identified the linear strain-specific and common epitopes of gonococcal pilin (11). By using synthetic peptides corresponding to different regions of the protein, we showed that antisera against pili from strain MS11 are predominantly strain specific and directed at two epitopes within a disulfide loop near the carboxyl terminus of the pilin molecule. We also determined that the sequence of this region varies greatly between different strains of gonococci. In contrast, the conserved, receptor-binding portion of the sequence (residues 31-92) contained only a weakly immunogenic epitope, common to all pili from the strains examined, between residues 48 and 60. However, when the central cyanogen bromide-generated fragment (CNBr II, residue...

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“…Initial expectations that a structure of the complex of concanavalin A and a saccharide would soon follow were not realized. Co-crystallization of concanavalin A and mannoside was reported by Hardman & Ainsworth (1976) but the crystals suffered from lattice disorders, which limited the resolution to 6 A. The saccharide-binding site was located, however.…”

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confidence: 99%

Refined structure of concanavalin A complexed with methyl α-D-mannopyranoside at 2.0 Å resolution and comparison with the saccharide-free structure

Naismith¹,

Emmerich²,

Habash³

et al. 1994

Acta Cryst D

152

136

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The three-dimensional structure of the complex between methyl a-D-mannopyranoside and concanavalin A has been refined at 2.0 A resolution. Diffraction data were recorded from a single crystal (space group P21212~, a = 123.7, b = 128.6, c = 67.2 A) using synchrotron radiation at a wavelength of 1.488 A. The final model has good geometry and an R factor of 19.9% for 58871 reflections (82% complete), within the resolution limits of 8 to 2 A, with F > 1.0tr(F). The asymmetric unit contains four protein subunits arranged as a dimer of dimers with approximate 222 point symmetry. Each monomer binds one saccharide molecule. Each sugar is bound to the protein by hydrogen bonds and van der Waals contacts. Although the four subunits are not crystallographically equivalent, the protein-saccharide interactions are nearly identical in each of the four binding sites. The differences that do occur between the four sites are in the structure of the water network which surrounds each saccharide; these networks are involved in crystal packing. The structure of the complex is compared with a refined saccharide-free concanavalin A structure. The saccharide-free structure is composed of crystallographically identical subunits, again assembled as a f To whom all correspondence should be addressed.(" 1994 International Union or" Crystallography Printed in Great Britain -all rights reserved dimer of dimers, but with exact 222 symmetry. In the saccharide complex the tetramer association is different in that the monomers tend to separate resulting in fewer intersubunit interactions. The average temperature factor of the mannoside complex is considerably higher than that of the saccharide-free protein. The binding site in the saccharide-free structure is occupied by three ordered water molecules and the side chain of Asp71 from a neighbouring molecule in the crystal. These occupy positions similar to those of the four saccharide hydroxyls which are hydrogen bonded to the site. Superposition of the saccharide-binding site from each structure shows that the major changes on binding involve expulsion of these ordered solvents and the reorientation of the side chain of Tyrl00. Overall the surface accessibility of the saccharide decreases from 370 to 100 A 2 when it binds to the protein. This work builds upon the earlier studies of Derewenda et al.

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Structure of the concanavalin A-methyl α-D-mannopyranoside complex at 6-Å resolution

Cited by 129 publications

References 28 publications

Peptide ligands for a sugar-binding protein isolated from a random peptide library.

Peptide ligands for a sugar-binding protein isolated from a random peptide library.

Antibodies to peptides corresponding to a conserved sequence of gonococcal pilins block bacterial adhesion.

Refined structure of concanavalin A complexed with methyl α-D-mannopyranoside at 2.0 Å resolution and comparison with the saccharide-free structure

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