Structural and functional consequences of peptide-carbohydrate mimicry. Crystal structure of a carbohydrate-mimicking peptide bound to concanavalin A

The functional consequences of peptide-carbohydrate mimicry were analyzed on the basis of the crystal structure of concanavalin A (ConA) in complex with a carbohydrate-mimicking peptide, DVFYPYPYASGS. The peptide binds to the non-crystallographically related monomers of two independent dimers of ConA in two different modes, in slightly different conformations, demonstrating structural adaptability in ConA-peptide recognition. In one mode, the peptide has maximum interactions with ConA, and in the other, it shows relatively fewer contacts within this site but significant contacts with the symmetry-related subunit. Neither of the peptide binding sites overlaps with the structurally characterized mannose and trimannose binding sites on ConA. Despite this, the functional mimicry between the peptide and carbohydrate ligands was evident. The peptide-inhibited ConA induced T cell proliferation in a dose-dependent manner. The effect of the designed analogs of the peptide on ConA-induced T cell proliferation and their recognition by the antibody response against alpha-d-mannopyranoside indicate a role for aromatic residues in functional mimicry. Although the functional mimicry was observed between the peptide and carbohydrate moieties, the crystal structure of the ConA-peptide complex revealed that the two peptide binding sites are independent of the methyl alpha-d-mannopyranoside binding site.     

Syntheses of two trimannose analogs each containing C-mannosyl or 5-thio-C-mannosyl residue: their affinities to concanavalin A

C-Mannosyl residue-containing trimannose ManC alpha(1,6)[Man alpha(1,3)Man] (2) and 5-thio-C mannosyl residue-containing trimannose 5SManC alpha(1,6)[Man alpha(1,3)Man] (3) were synthesized via a glycosyl radical addition to enone derivative of mannose (6). Dissociation constants for the binding of these trisaccharides to concanavalin A (ConA) were determined by a fluorescence anisotropy inhibition assay: Kd = 198 and 31 microM, respectively. The unexpectedly large Kd value for the compound 2 compared with the compound 3 and the natural trimannose 1 demonstrates a characteristic of C-glycoside.     

A monosaccharide-modified peptide phage library for screening of ligands to carbohydrate-binding proteins

A monosaccharide-modified β-loop peptide library displayed on phage has been constructed and used for the screening of glycopeptide ligands against a carbohydrate-binding protein. The β-loop peptide library was designed and modified with a mannose derivative on phage. The glycopeptide ligands to concanavalin A (ConA), a mannose-binding protein, were obtained from the mannose-modified peptide phage library. The amino acids neighboring the mannose unit of glycopeptides not only reinforced the binding affinity but also gave diverse binding characteristics.     

Enhanced binding of a rationally designed peptide ligand of concanavalin a arises from improved geometrical complementarity

The structural basis of affinity enhancement was addressed by analyzing the interactions between concanavalin A and the carbohydrate-mimicking peptide ligands. Based on the crystal structures of concanavalin A in complex with these peptides [Jain, D., Kaur, K. J., Sundaravadivel, B., and Salunke, D. M. (2000) J. Biol. Chem. 275, 16098-16102; Jain, D., Kaur, K. J., and Salunke, D. M. (2001) Biophys. J. 80, 2912-2921], a high-affinity analogue was designed. This analogue (acetyl-MYWYPY-amide) binds to the lectin with 32-fold enhanced affinity compared to the corresponding precursor peptides. The crystal structure of concanavalin A bound to the designed peptide has been determined. A peptide molecule binds to each of the crystallographically independent monomers of the tetrameric lectin. The four bound peptide molecules exhibit two major conformations both of which are extended. Unlike in the case of other concanavalin A binding peptides, the structural variations within different conformers of this analogue are marginal. It is apparent that the deletion of the structurally variable region of the larger peptides has led to an improved complementarity and increased buried surface area in the case of the designed peptide. The crystal structure also showed the formation of two backbone hydrogen bonds between the ligand and the ligate which were not present in the complexes of the precursor peptides. The observed structural features adequately explain the enhanced binding of the designed analogue.     

A comparison of direct and indirect techniques using ferritin-conjugated ligands for the localization of concanavalin A binding sites on isolated hepatocyte plasma membranes

Concanavalin A binding sites have been localized on isolated plasma membranes both by a direct technique involving ferritin-concanavalin A and by an indirect technique in which membranes were treated successively with concanavalin A, rabbit anti-concanavalin A, and ferritin-conjugated sheep anti-rabbit F(ab')2. Binding studies showed that, at saturation, less than 25% of the concanavalin A binding sites were accessible to ferritin-concanavalin A. The decreased binding was apparently related to steric factors, since membranes saturated with the conjugated ligand were able to bind additional concanavalin A, and since the conjugated ligand, once bound to the membrane, caused the same inhibition of the membrane-bound enzyme 5'-nucleotidase as concanavalin A. Nonspecific binding sites accounted for 10% of the total binding of ferritin-concanavalin A and were localized mainly on the cytoplasmic side of the membrane, whereas specific sites were on the external side. The indirect technique, which was expected to increase the binding of ferritin-conjugate to the membrane, resulted in the binding of ferritin to less than 15% of the concanavalin A binding sites, and did not decrease the nonspecific binding.     

Crystallographic structure of metal-free concanavalin A at 2.5 Å resolution

The three-dimensional structure of demetallized concanavalin A has been determined at 2.5 Å resolution and refined to a crystallographic R-factor of 18%. The lectin activity of concanavalin A requires the binding of both a transition metal ion, generally Mn²⁺, and a Ca²⁺ ion in two neighboring sites in close proximity to the carbohydrate binding site. Large structural differences between the native and the metal-free lectin are observed in the metal-binding region and consequently for the residues involved in the specific binding of saccharides. The demetallization invokes a series of conformational changes in the protein backbone, apparently initiated mainly by the loss of the calcium ion. Most of the Mn²⁺ ligands retain their position, but the Ca²⁺ binding site is destroyed. The Ala207-Asp208 peptide bond, in the β-strand neighboring the metal-binding sites, undergoes a cis to trans isomerization. The cis conformation for this bond is a highly conserved feature among the leguminous lectins and is critically maintained by the Ca²⁺ ion in metal-bound concanavalin A. A further and major change adjacent to the isomerized bond is an expansion of the loop containing the monosaccharide ligand residues Leu99 and Tyr100. The dispersion of the ligand residues for the monosaccharide binding site (Asn14, Agr228, Asp208, Leu99, and Tyr100) in metalfree concanavalin A abolishes the lectin's ability to bind saccharides. Since the quaternary structure of legume lectins is essential to their biological role, the tetramer formation was analyzed. In the crystal (pH 5), the metal-free concanavalin A dimers associate into a tetramer that is similar to the native one, but with a drastically reduced number of inter-dimer interactions. This explains the tetramer dissociation into dimers below pH values of 6.5. © 1995 Wiley-Liss, Inc.     

Analysis of lectin-specific cell surface glycoprotein on neuroblastoma cells

The binding sites for the lectins wheat germ agglutinin, Ricinus communis agglutinin and concanavalin A on mouse neuroblastoma cell membranes were identified using SDS-gel electrophoresis in combination with fluorescent lectins. Ricinus communis agglutinin and wheat germ agglutinin were found to bind almost exclusively to a single polypeptide with an apparent molecular weight of 30 000. Concanavalin A labeled over 20 different polypeptides, most with molecular weights greater than 50 000. However, when the neuroblastoma cells were treated with concanavalin A so as to internalize all the concanavalin A binding sites visible at the level of the fluorescent microscope and the purified plasma membranes analyzed for their concanavalin A binding polypeptides, only four of the 20 glycopolypeptides were missing or significantly reduced in amount. Thus, these four high molecular weight concanavalin A-binding polypeptides appear to be the major cell surface receptors for concanavalin A. Binding studies with iodinated concanavalin A indicated that these polypeptides represented the high affinity concanavalin A binding sites $\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 2 · 10}{}^{\mathrm{?7}}\text{M}$). Low affinity concanavalin A binding sites were present on the cell surface after internalization of high affinity concanavalin A binding sites.     

Binding of Concanavalin A and its effect on the differentiation of Dictyostelium Discoideum.

Dictyostelium discoideum amoebae have two classes of binding sites for concanavalin A, one accessible to a competitive inhibitor of concanavalin A binding and one inaccessible. Uptake of concanavalin A occurs and is associated with a transfer from accessible to inaccessible binding sites. Low amounts of concanavalin A bound to cells produce a slight stimulation of differentiation, and this is associated with an increase in production of phosphodiesterase. Above a critical amount of concanavalin A bound, cell differentiation and phosphodiesterase production are inhibited. The relationship of concanavalin A-induced membrane changes to cell differentation is discussed.     

The dual-function disabled-1 PTB domain exhibits site independence in binding phosphoinositide and peptide ligands

While typical intracellular protein modules have only one ligand-binding site, there are rare examples of single modules that bind two different ligands at distinct binding sites. Here we present a detailed mutational and energetic analysis of one such domain, the phosphotyrosine binding (PTB) domain of Disabled-1 (Dab1), which binds to both peptide and phosphoinositide (PI) ligands simultaneously at structurally distinct binding sites. Through the techniques of isothermal titration calorimetry (ITC), analysis of Dab1 PTB domain mutants, and nuclear magnetic resonance (NMR), we have evaluated the characteristics of binding of the Dab1 PTB domain to various peptide and PI ligands. These studies reveal that the presence of saturating concentrations of one ligand has little effect on the binding constant for a second ligand at the other site. In addition, proteins with single-point mutations in the peptide-binding site retain native affinity for PI ligands, while proteins with mutations that prevent PI binding retain native affinity for peptide. NMR titrations show that the final structure of the ternary complex is the same independent of the order of addition of the two ligands. Together, these studies show that binding of peptide and PI ligands is energetically independent and noncooperative.     

Concanavalin A, a receptor protein with apparently co-operative binding characteristics.

Laser nephelometry is a suitable technique for the quantitative determination and differentiation of both lectins and glycoconjugates in the low-picomolar range. Simultaneously this method renders possible investigations on the specificity and mode of interaction between lectins and different ligands. The results demonstrate that the degree of co-operativity between concanavalin A and the respective glycoconjugate is dependent on the presence of hydrophobic binding sites and can be substantially altered by conformational changes of the ligand. The transition from apotransferrin to Fe3+-transferrin induces a transformation of the sigmoidal-shaped binding curve to a hyperbolic one. Hence, at low concentrations, Fe3+-transferrin is bound far better than apotransferrin, whereas maximal binding is nearly identical. After removal of N-acetylneuraminate, concanavalin A is less efficient in differentiating between the Fe3+-charged and Fe3+-free (apo) forms of transferrin.     

Multivalent protein binding in carbohydrate-functionalized monolayers through protein-directed rearrangement and reorientation of glycolipids at the air-water interface

Multivalent protein binding plays an important role not only in biological recognition but also in biosensor preparation. Infrared reflection absorption spectroscopy and surface plasmon resonance techniques have been used to investigate concanavalin A (Con A) binding to binary monolayers composed of 1,2-di-O-hexadecyl-sn-glycerol and derived glycolipids with the mannose moieties. The glycolipids in the binary monolayers at the air-water interface underwent both lateral rearrangement and molecular reorientation directed by Con A in the subphase favorable to access of the carbohydrate ligands to protein binding pockets for the formation of multivalent binding sites and the minimization of steric crowding of neighboring ligands for enhanced binding. The amounts of specifically bound proteins in the binary monolayers at the air-water interface were accordingly increased in comparison with those in the initially immobilized monolayers at the air-water interface. The directed rearranged binary monolayers with multivalent protein binding were preserved for the preparation of biosensors.     

Determining binding sites of polycyclic aromatic small molecule-based amyloid-beta peptide aggregation modulators using sequence-specific antibodies

Numerous aromatic small molecule modulators of amyloid-beta peptide (Aβ) monomer aggregation and neurotoxicity have been identified with the ultimate goal of Alzheimer’s disease (AD) treatment. Determining binding sites of these modulators on Aβ monomer is an important topic in the mechanistic understanding of AD pathology and drug development. However, Aβ monomer binding sites have been reported for only a very limited number of Aβ modulators. In this article, we present a convenient method for determining aggregation-modulating polycyclic aromatic small molecule ligand binding sites on Aβ monomer using immunostaining with a panel of Aβ sequence-specific antibodies. To validate our technique, we first examined one modulating aromatic ligand, Congo Red, with known binding sites, which yielded consistent results with previous findings. Then, using the same technique, binding sites on Aβ of four known Aβ monomer aggregation modulators, Erythrosin B, Eosin Y, Phloxine B, and Rose Bengal, were determined. The identified ligand binding sites were also confirmed by a separate fluorescence quenching-based assay using a panel of overlapping Aβ sub-fragments. The technique described here greatly increases researchers’ ability to determine the Aβ monomer binding site(s) of aggregation-modulating aromatic small molecule ligands and to screen for new ligands that bind specific regions on Aβ.     

Designing cyclopentapeptide inhibitor as potential antiviral drug for dengue virus ns5 methyltransferase

NS5 methyltransferase (Mtase) has a crucial role in the replication of dengue virus. There are two active sites on NS5 Mtase i.e., SAM and RNA-cap binding sites. Inhibition of the NS5 Mtase activity is expected to prevent the propagation of dengue virus. This study was conducted to design cyclic peptide ligands as enzyme inhibitors of dengue virus NS5 Mtase through computational approach. Cyclopentapeptides were designed as ligand of SAM binding site as much as 1635 and 736 cyclopentpeptides were designed as ligand of RNA-cap binding site. Interaction between ligand and NS5 Mtase has been conducted on the Docking simulation. The result shows that cyclopentapeptide CTWYC was the best peptide candidate on SAM binding site, with estimated free binding energy -30.72 kca/mol. Cyclopentapeptide CYEFC was the best peptide on RNA-cap binding site with estimated free binding energy -22.89 kcal/mol. Both peptides did not have tendency toward toxicity properties. So it is expected that both CTWYC and CYEFC ligands could be used as a potential antiviral drug candidates, which can inhibit the SAM and RNA-cap binding sites of dengue virus NS5 Mtase.     

Flow cytometric measurement of fluorescence resonance energy transfer on cell surfaces. Quantitative evaluation of the transfer efficiency on a cell-by-cell basis

A method has been developed for the determination of the efficiency (E) of the fluorescence resonance energy transfer between moieties on cell surfaces by use of a computer-controlled flow cytometer capable of dual wavelength excitation. The absolute value of E may be calculated on a single-cell basis. The analysis requires the measurement of samples stained with donor and acceptor conjugated ligands alone as well as together. In model experiments HK 22 murine lymphoma cells labeled with fluorescein-conjugated concanavalin A (Con A) and/or rhodamine conjugated Con A were used to determine energy transfer histograms. Using the analytic solution to energy transfer in two dimensions, a high surface density of Con A binding sites was found that suggests that the Con A receptor sites on the cell surface are to a degree preclustered . We call this technique flow cytometric energy transfer ( FCET ).     

Location and nature of the residues important for ligand recognition in G-protein coupled receptors

The overall structure of the biogenic amine subclass of the G-protein-coupled receptors, and of their ligand binding sites, is discussed with the aim of highlighting the major structural features of these receptors that are responsible for ligand recognition. A comparison is made between biogenic amine receptors, peptide receptors of the rhodopsin class, and the secretin receptors which all have peptide ligands. The question of where the peptide ligands bind, whether at extracellular sites or within the transmembrane helix bundle, is discussed. The suitability of the rhodopsin crystal structure as a template for construction of homology models is discussed and it is concluded that there are many reasons why a caution should be issued against using it uncritically.     

Antibodies against preselected peptides to map functional sites on the acetylcholine receptor

Rabbit immune sera and mouse monoclonal antibodies were raised against the synthetic peptide Tyr-Cys-Glu-Ile-Ile-Val matching in sequence residues 127-132 of the alpha-subunit of all nicotinic acetylcholine receptors sequenced so far. Representative cholinergic ligands did not interfere with the binding of these antibodies to the receptor from Torpedo marmorata, indicating that this sequence is not part of the binding sites for cholinergic ligands. The applicability of antigenic sites analysis to the mapping of functional sites on receptor proteins is discussed.     

Collisional unfolding of multiprotein complexes reveals cooperative stabilization upon ligand binding

Cooperative binding mechanisms are a common feature in biology, enabling a diverse range of protein-based molecular machines to regulate activities ranging from oxygen uptake to cellular membrane transport. Much, however, is not known about such cooperative binding mechanisms, including how such events typically add to the overall stability of such protein systems. Measurements of such cooperative stabilization events are challenging, as they require the separation and resolution of individual protein complex bound states within a mixture of potential stoichiometries to individually assess protein stabilities. Here, we report ion mobility-mass spectrometry results for the concanavalin A tetramer bound to a range of polysaccharide ligands. We use collision induced unfolding, a relatively new methodology that functions as a gas-phase analog of calorimetry experiments in solution, to individually assess the stabilities of concanavalin A bound states. By comparing the differences in activation voltage required to unfold different concanavalin A–ligand stoichiometries, we find evidence suggesting a cooperative stabilization of concanavalin A occurs upon binding most carbohydrate ligands. We critically evaluate this observation by assessing a broad range of ligands, evaluating the unfolding properties of multiple protein charge states, and by comparing our gas-phase results with those obtained from calorimetry experiments carried out in solution.     

Analysis of sequence variation among legume lectins. A ring of hypervariable residues forms the perimeter of the carbohydrate-binding site.

Twelve plant lectins from the Papilionoideae subfamily were selected to represent a range of carbohydrate specificities, and their sequences were aligned. Two variability indices were applied to the aligned sequences and the results were analysed using the three-dimensional structures of concanavalin A and the pea lectin. The areas of greatest variability were located in the carbohydrate-binding site region, forming a perimeter around a well-conserved core. These residues are inferred to be specificity determining, in the manner of antibodies, and the most variable position corresponded to Tyr100 in concanavalin A, a known ligand contact residue. In addition to the five peptide loops known to form the binding site from crystallographic studies, a sixth segment with variable residues was located in the binding-site region, and this may contribute to oligosaccharide specificity. In their overall composition, the lectin sites resemble those of the sugar-transport proteins rather than antibodies. The prospects for modelling lectin binding sites by the methods used for antibodies were also assessed.     

Changes in the distribution of platelet membrane proteins revealed by energy transfer

The redistribution of platelet membrane proteins in response to platelet activation was studied. To investigate this process we prepared a variety of platelet ligands, including di- and tetrameric concanavalin A, IgG, thrombin, wheat-germ agglutinin and other lectins. These ligands were conjugated either with acceptor (rhodamine isothiocyanate) or donor (fluoresceine isothiocyanate) fluorophore. Platelets exposed to various combinations of ligand species were stimulated with different aggregating agents, and changes in sensitized fluorescence emission or donor quenching were recorded. Energy transfer was observed with thrombin, dimeric concanavalin A after addition of thrombin and various combinations of dimeric concanavalin A with other membrane ligands. The preincubation of platelets with colchicine prevented energy transfer between appropriate ligand pairs and platelet activator. Our studies show that nonradiative energy transfer can be used to analyze redistribution of membrane receptor sites in platelets.     

Relationship of prolactin receptors to concanavalin A binding.

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of 125I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar alpha-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 mu/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (Kd) as the controls. The binding of 125I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (Kd approximately equal to 6.6 . 10(-8) M. At high lectin concentrations, low affinity (Kd approximately equal to 6.7 . 10(-5) M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 microgram/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone. Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized 125I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by alpha-methyl-D-mannopyranoside.