The Modes of binding of carbohydrates to concanavalin a : a computer modelling approach

Abstract

Concanavalin A (Con A), the carbohydrate?binding protein (lectin) originally isolated from the jack bean (Canavalia ensiformis), specifically binds to carbohydrates containing ??D?mannose and ??D?glucose residues in non?reducing terminal positions. It can also recognize certain oligosaccharide sequences lacking terminal mannosyl or glucosyl groups, making it a widely useful reagent in glycobiology. The carbohydrate?binding specificity of Con A and related lectins has greatly contributed to advances in studying cell surface carbohydrates, including the isolation, characterization, and sequencing of polysaccharides and glycoproteins, and the elucidation of important changes in structure, organization, and dynamics of cell?surface molecules during development, virus infection, oncogenic transformation, cell growth, cell movement, and cell–cell interactions.To appreciate the use and role of Con A in cell surface research, an understanding of the three?dimensional structures of Con A–carbohydrate complexes is essential. X?ray diffraction is the most powerful method available to determine the three?dimensional structures of macromolecules and their complexes at atomic resolution. Although the structure of Con A was first reported as early as the 1970s, detailed knowledge about the effects of carbohydrate configuration, substitution, and linkage on binding modes to this lectin remains incomplete.

Furthermore, data obtained on Con A–sugar interactions from spectroscopic and solution studies have sometimes been in disagreement about (1) the size and shape of the carbohydrate binding site on Con A, (2) the sites of attachment on the oligosaccharide, (3) the residues on Con A responsible for binding carbohydrates, and (4) the reasons for differences in binding affinities of Con A to various sugars.

Hence, attempts have been made, using available X?ray crystallographic data, to study the modes of carbohydrate binding to Con A by computer modelling techniques to obtain information regarding these aspects. Such information is important not only for understanding the mechanism of carbohydrate binding to Con A but also the role of Con A in cell surface research.

This thesis contains eight chapters. The first chapter deals with a brief review of the structure and specificity of Con A relevant to the present study. In Chapter II, the nomenclature of sugars and conformational parameters are presented, along with various potential energy functions used in the present work. The procedure followed for the computer modelling of the protein–ligand interactions is also outlined.

Chapter III deals with the probable modes of binding of methyl??? and methyl???D?glucopyranosides and some of their derivatives to Con A, as determined from computer modelling studies. Methyl???D?glucopyranoside can bind to Con A in one mode, whereas methyl???D?glucopyranoside can bind in several modes. In the most favorable binding poses, methyl???D?glucopyranoside binds in a different orientation compared to methyl???D?glucopyranoside. Substitution at certain ring positions (e.g., C?3 or C?4) drastically affects allowable binding orientations and binding affinity, whereas substitution at the C?2 position does not significantly change the allowed orientations in the Con A binding site. These models help interpret NMR and kinetic data on sugar binding.

The probable modes of binding of methyl???D?mannopyranoside, methyl???D?mannopyranoside, 2?O?methyl???D?mannopyranose, methyl?2?O?methyl???D?mannopyranoside, and methyl???D?N?acetylmannosamine to Con A are presented in Chapter IV. All sugars, except methyl???D?N?acetylmannosamine, can reach the binding site of Con A with a restricted number of binding orientations. Methyl?2?O?methyl???D?mannopyranoside binds in one mode, whereas the other sugars can adopt multiple binding modes. The high potency of methyl???D?mannopyranoside compared to methyl???D?mannopyranoside is mainly due to favorable hydrophobic interactions with key binding residues like Leu(99) and Tyr(100), as well as the formation of hydrogen bonds with the protein.Chapters V and VI describe the modes of binding of ?? and ??glycosidically linked disaccharides to Con A. In general, ??linked disaccharides can reach the binding site in their most probable low?energy conformers, whereas ??linked disaccharides often must adopt higher?energy conformations to bind. However, ?(1?2)?linked sugars are an exception and can be accommodated in multiple favorable orientations due to an extended binding site that can engage more than one mannose moiety.In Chapter VII, the preferred conformations of some oligosaccharides having the trimannosyl core (Man?1?6[Man?1?3]Man) that interact with Con A are presented. In the minimum energy conformers of the trimannosidic core, the mannose residue on the ?(1?6) arm comes close to one of the N?acetylglucosamine residues of the core. Interactions between these residues help determine the preferred orientations of both the core and the ?(1?6) arm. The addition of N?acetylglucosamine to terminal residues does not alter the preferred core conformers but changes some higher energy conformations. The minimum energy conformer broadly agrees with available X?ray data on Con A complexes.In Chapter VIII, the probable binding modes of these carbohydrates are presented. These studies suggest that Con A binds preferentially to terminal mannose residues of the trimannosidic core and less strongly to internal mannose or terminal N?acetylglucosamine residues in higher?energy conformations. Oligosaccharides with terminal mannose residues form more hydrogen bonds with Con A than those without. The differential binding affinities of various oligosaccharides largely depend on the availability of binding?competent conformers and on specific non?covalent interactions between the sugar and the protein.

In conclusion, the present study provides valuable information about (a) the modes of binding of various sugars to Con A, (b) the effect of carbohydrate configuration, substitution, and linkage on sugar binding to Con A, and (c) the size and nature of the binding site, all of which are important for better understanding Con A as a tool in biological research.