V. S. R. Rao

Theoretical studies on the conformation of monosialogangliosides and disialogangliosides

The preferred conformation of gangliosides GM3, GM2, GM1, GD1a and GD1b have been studied by computing their potential energies. The conformation of NeuNAc in GM3 differs from that expected for the same residue in GM2 and GM1. The NeuNAc residues in GM2 and GM1 exhibit identical conformations. Theory predicts that the terminal NeuNAc of GD1a is conformationally similar to that of GM3 and that the internal one is similar in conformation to those present in GM2 and GM1 in agreement with NMR studies. The differences in chemical shifts of the C2 and C3 carbons of the internal and terminal NeuNAc of GD1a have been attributed to differences in orientation. The present studies suggest that the binding site of cholera toxin is much smaller than that of tetanus toxin. The preferred shape of these gangliosides correlate well with their biological properties.

Architecture of the sugar binding sites in carbohydrate binding proteins—a computer modeling study

Different sugars, Gal, GalNAc and Man were docked at the monosaccharide binding sites of Erythrina corallodenron (EcorL), peanut lectin (PNA), Lathyrus ochrus (LOLI), and pea lectin (PSL). To study the lectin-carbohydrate interactions, in the complexes, the hydroxymethyl group in Man and Gal favors, gg and gt conformations respectively, and is the dominant recognition determination. The monosaccharide binding site in lectins that are specific to Gal/GalNAc is wider due to the additional amino acid residues in loop D as compared to that in lectins specific to Man/Glc, and affects the hydrogen bonds of the sugar involving residues from loop D, but not its orientation in the binding site. The invariant amino acid residues Asp from loop A, and Asn and an aromatic residue (Phe or Tyr) in loop C provides the basic architecture to recognize the common features in C4 epimers. The invariant Gly in loop B together with one or two residues in the variable region of loop D/A holds the sugar tightly at both ends. Loss of any one of these hydrogen bonds leads to weak interaction. While the subtle variations in the sequence and conformation of peptide fragment that resulted due to the size and location of gaps present in amino acid sequence in the neighborhood of the sugar binding site of loop D/A seems to discriminate the binding of sugars which differ at C4 atom (galacto and gluco configurations). The variations at loop B are important in discriminating Gal and GalNAc binding. The present study thus provides a structural basis for the observed specificities of legume lectins which uses the same four invariant residues for binding. These studies also bring out the information that is important for the design/engineering of proteins with the desired carbohydrate specificity.

Molecular modeling studies on binding of bFGF to heparin and its receptor FGFR1.

Sugar induced protein-protein interactions play an important role in several biological processes. The carbohydrate moieties of proteoglycans, the glycosaminoglycans, bind to growth factors with a high degree of specificity and induce interactions with growth factor receptors, thereby regulate the growth factor activity. We have used molecular modeling method to study the modes of binding of heparin or heparan sulfate proteoglycans (HSPGs) to bFGF that leads to the dimerization of FGF receptor 1 (FGFR1) and activation of receptor tyrosine kinase. Homology model of FGFR1 Ig D(II)-D(III) domains was built to investigate the interactions between heparin, bFGF and FGFR1. The structural requirements to bridge the two monomeric bFGF molecules by heparin or HSPGs and to simulate the dimerization and activation of FGFR1 have been examined. A structural model of the biologically functional dimeric bFGF-heparin complex is proposed based on: (a) the stability of dimeric complex, (b) the favorable binding energies between heparin and bFGF molecules, and (c) its accessibility to FGFR1. The modeled complex between heparin, bFGF and FGFR1 has a stoichiometry of 1 heparin: 2 bFGF: 2 FGFR1. The structural properties of the proposed model of bFGF/heparin/FGFR1 complex are consistent with the binding mechanism of FGF to its receptor, the receptor dimerization, and the reported site-specific mutagenesis and biochemical cross-linking data. In the proposed model heparin bridges the two bFGF monomers in a specific orientation and the resulting complex induces FGF receptor dimerization, suggesting that in the oligosaccharide induced recognition process sugars orient the molecules in a way that brings about specific protein-protein or protein-carbohydrate interactions.

Three dimensional structure of the soybean agglutinin Gal/GalNAc complexes by homology modeling.

Complexes of soybean agglutinin (SBA) with galactose (Gal) and N-acetyl galactosamine (GalNAc) have been modeled based on its homology to erythrina corallodendron (EcorL) lectin. The three dimensional structure of SBA-Gal modeled with homology techniques agrees well with SBA-(beta-LacNAc)2Gal-R complex determined by X-ray crystallographic techniques at the beta-sheet regions and the regions where Ca2+ and Mn2+ ions bind. However, significant deviations have been observed between the modeled and the X-ray structures, particularly at the loop regions where the polypeptide chain could not be unequivocally traced in the X-ray structure. The hydrogen bonding scheme, predicted from the homology model, shows that the invariant residues i.e. Asp, Gly, Asn, and aromatic residues (Phe) found in all other legume lectins, bind Gal, slightly in a different way than reported in X-ray structure of SBA-pentasaccharide complex. The higher binding affinity of GalNAc over Gal to SBA is due to additional hydrophobic interactions with Tyr107 rather than a hydrogen bond between N-acetamide group of the sugar and the side chain of Asp88 as suggested from X-ray crystal structure studies. Our modeling also suggest that the variation in the length of the loop D observed among galactose binding legume lectins may not have any effect on the binding of sugar at the monosaccharide specific site of the lectins. Soybean agglutinin (SBA) is a member of the leguminous family of lectins. They generally possess a single carbohydrate binding site, besides the tightly bound Ca2+ and Mn2+ ions which are required for their carbohydrate binding activity. They possess a high degree of sequence homology and about 50% of the amino acid residues are invariant. Some of these invariant amino acid residues are involved in the binding of sugar moieties and in metal ion coordination. X-ray crystallographic studies showed that their three-dimensional structures are very similar, though they differ in their carbohydrate binding specificity (1-6). Three of the invariant residues Asp, Gly, and Asn, besides an aromatic residue (Phe or Tyr), are involved in carbohydrate binding. Independent of their sugar specificity, these four residues in legume lectins provide the basic frame for the sugar to bind.

Complex carbohydrates: 2. The modes of binding of complex carbohydrates to concanavalin A — a computer modelling approach

The probable modes of binding of some complex carbohydrates, which have the trimannosidic core structure (Man3GlcNAc2), to concanavalin A (Con A) have been determined using a computer modelling technique. These studies show that Con a can bind to the terminal mannose residues of the trimannosidic core structure and to the internal mannosyl as well as to the terminal N-acetylglucosamine residues of the N-acetylglucosamine substituted trimannosidic core structure. The oligosaccharide with terminal mannose residues can bind in its minimum energy conformers, whereas the oligosaccharide with internal mannosyl and terminal N-acetylglucosamine residues can bind only in higher energy conformers. In addition the former oligosaccharide forms more hydrogen bonds with Con A than the latter. These results suggest that, for these oligosaccharides, the terminal mannose residue has a much higher probability of reaching the binding site than either the internal mannosyl or the terminal N-acetylglucosamine residues. The substitution of a bisecting N-acetylglucosamine residue on these oligosaccharides, affects significantly the accessibility of the residues which bind to Con A and thereby reduces their binding affinity. It thus seems that the binding affinity of an oligosaccharide to Con A depends not only on the number of sugar residues which possess free 3-, 4- and 6-hydroxyl groups but also on the accessibility of these sugar residues to Con A. This study also reveals that the sugar binding site of Con A is small and that the interactions between Con A and carbohydrates are extended slightly beyond the single sugar residue that is placed in the binding site.

Complex carbohydrates: 1. Conformational studies on some oligosaccharides related to N-glycosyl proteins which interact with concanavalin A

Empirical potential energy calculations have been carried out to determine the preferred conformations of some oligosaccharides having the trimannosidic core structure (Man3GlcNAc2) and which interact with concanavalin A. In the minimum energy conformations for the trimannosidic core the mannose residue on the Man α(1–6) arm comes close to one of the N-acetylglucosamine residues of the core. The addition of N-acetylglucosamine residues to the terminal mannose residues does not alter the preferred conformation of the trimannosidic core although it alters the relative preference of some of the higher energy conformations. The minimum energy conformation broadly agrees with available X-ray data. The presence of a bisecting N-acetylglucosamine residue on the middle mannose does not push the trimannosidic core to any new conformation but it does alter the relative preference for a particular conformation.

The modes of binding methyl-alpha (and beta)-D-glucopyranosides and some of their derivatives to concanavalin A--a theoretical approach

The probable modes of binding of Methyl--alpha (and beta)-D-glucopyranosides and some of their derivatives to concanavalin A have been proposed from theoretical studies. Theory predicts that beta-MeGlcP can bind to ConA in three different modes whereas alpha-MeGlcP can bind only in one mode. beta-MeGlcP in its most favourable mode of binding differs from alpha-MeGlcP in its alignment in the active-site of the lectin where it binds in a flipped or inverted orientation. Methyl substitution at the C-2 atom of the alpha-MeGlcP does not significantly affect the possible orientations of the sugar in the active-site of the lectin. Methyl substitution at C-3 or C-4, however, affects the allowed orientations drastically leading to the poor inhibiting power of Methyl-3-O-methyl-alpha-D-glucopyranoside and the inactivity of Methyl-4-O-methyl-alpha-D-glycopyranoside. These studies suggest that the increased activity of the alpha-MeGlcP over beta-MeGlcP may be due to the possibility of formation of better hydrogen bonds and to hydrophobic interactions rather than to steric factors as suggested by earlier workers. These models explain the available NMR and other binding studies.

Theoretical studies on the conformations of higher gangliosides

The possible conformations of higher gangliosides (GD3, GT1a. GT1b, GQ1b) have been determined by computing their potential energy using semi-empirical potential functions. The favoured conformation of the disialic acid fragment in these gangliosides is independent of its position (internal or terminal). The favoured conformations of these gangliosides have also been correlated to their biological activity. The results suggest that tetanus toxin and sendai virus may have a large binding site which can accommodate at least four sugar residues.

Conformational studies on the ABH and Lewis blood group oligosaccharides

The possible conformations for the ABH and Lewis blood group oligosaccharides have been studied by an energy-minimisation procedure using empirical potential functions. It has been found that the conformation of the core structure is not altered significantly by the addition of l-fucose, galactose or N-acetyl galactosamine residues at the non-reducing end. Correlation of the preferred conformations with their known binding properties suggests that the differences between type 1 and type 2 structures become significant only when a large enough fragment of the determinant is considered. It is suggested that non-specific reagents may have small binding sites while the reagents that are specific for type 1 or type 2 structures may have larger binding sites. A two-pocket model has been proposed for antibodies and lectins which can distinguish the A1 and A2 antigens.

Computer simulation of protein—carbohydrate complexes: application to arabinose-binding protein and pea lectin

The CCEM method (Contact Criteria and Energy Minimisation) has been developed and applied to study protein-carbohydrate interactions. The method uses available X-ray data even on the native protein at low resolution (above 2.4 A) to generate realistic models of a variety of proteins with various ligands.The two examples discussed in this paper are arabinose-binding protein (ABP) and pea lectin. The X-ray crystal structure data reported on ABP-β-l-arabinose complex at 2.8, 2.4 and 1.7 A resolution differ drastically in predicting the nature of the interactions between the protein and ligand. It is shown that, using the data at 2.4 A resolution, the CCEM method generates complexes which are as good as the higher (1.7 A) resolution data. The CCEM method predicts some of the important hydrogen bonds between the ligand and the protein which are missing in the interpretation of the X-ray data at 2.4 A resolution. The theoretically predicted hydrogen bonds are in good agreement with those reported at 1.7 A resolution. Pea lectin has been solved only in the native form at 3 A resolution. Application of the CCEM method also enables us to generate complexes of pea lectin with methyl-α-d-glucopyranoside and methyl-2,3-dimethyl-α-d-glucopyranoside which explain well the available experimental data in solution.