Atomic Basis of Protein–Carbohydrate Interactions: An Overview

NATHAN SHARON

Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel

1 Introduction

Protein–carbohydrate interactions are the basis of numerous biological processes, both normal and pathological ones. They include the enzymatic synthesis and degradation of oligo- and polysaccharides, intracellular sorting of glycoconjugates, transport of carbohydrates into living cells and of their derivatives into subcellular organelles, the immunological response to carbohydrate antigens, and migration of leukocytes to sites of inflammation. These interactions also play a key role in a variety of cell adhesion phenomena, among them the attachment of parasites, fungi, bacteria, and viruses to host cells, the first step in the initiation of infection. The high selectivity required for this attachment, as well as the binding of a variety of microbial toxins to cells, is provided by a specific stereochemical fit between complementary molecules, one a carrier of biological information (such as complex carbohydrates) and the other capable of decoding such information (carbohydrate-binding proteins, belonging to the class of lee tins). This concept has its origins in the lock-and-key hypothesis, introduced by Emil Fisher at the end of the 19th century to explain the specificity of interactions between enzymes and their substrates, i.e., between molecules in solution; it was subsequently extended to describe the interactions of cells with soluble molecules and with other cells.

Complex carbohydrates are commonly found at the cell surface, where they are positioned to interact with suitable proteinaceous receptors, primarily lee tins, in solution or on the surfaces of other cells. These proteins, originally identified as sugar-specific hemagglutinins, are currently known as cell-recognition molecules. They are geared to distinguish between different oligosaccharides, whether as such or as part of glycoconjugates (primarily glycoproteins and glycolipids). Like other carbohydrate-binding proteins, whether enzymes, anticarbohydrate antibodies, or sugar transporters, lectins are structurally diverse, differing markedly in size, tertiary and quaternary structure, as well as the structure of their combining sites.

2 Combining Sites

A major source of information about the combining sites of lectins is the X-ray crystallography study of their complexes with ligands. By now, the structures of close to 200 lectins, and over 300 of their complexes with carbohydrates, have been solved largely by this method (www.cermav.cnrs.fr/lectines/); most of those from bacterial or viral sources are listed in Table 1. Other inputs include binding experiments with sugars and their derivatives, site-directed mutagenesis, and, to a limited extent, also NMR experiments and molecular modeling. Such studies have shown that like the lectins themselves, the sites are diverse, even when their specificity is the same, although within a given lectin family the sites may be similar. The sites appear to be preformed, since few conformational changes occur upon ligand binding. They are mostly in the form of shallow depressions on the surface of the protein, where typically only one or two edges or faces of the ligand are bound, and are thus similar to those of anticarbohydrate antibodies or glycosidases. In a few lectins, the combining sites are in the form of deep clefts.

The participation of a particular amino acid of a lectin and of a specific group of the carbohydrate ligand in the interaction between the two can be assessed by different methods. In the case of the amino acids, it is done mainly by site-directed mutagenesis, as mentioned earlier. This technique, combined with ligand-binding experiments, also provides information on the relative contribution of individual residues to the protein-carbohydrate interaction. As to the carbohydrate ligand, its hydrogen bonding pattern in the complex can be mapped by studies with deoxy-, methoxy-, and deoxyfluoro sugar analogs. It is only X-ray crystallography, however, that provides detailed information at the atomic level on the interplay between the two molecules. Still, it should be kept in mind that a crystal represents a frozen constellation often obtained from a concentrated solution in a nonphysiological milieu, without any indication on the status of the involved molecules prior to complex formation. The use of nonphysiological crystallization conditions may induce conformational changes in the protein and alter the mode of binding. Therefore, although the value of X-ray crystallography is not disputable, it is essential to complement the data obtained by this method with information from other sources (e.g., NMR) on the solution structure of the lectin-ligand complex.

3 Lectin–Carbohydrate Bonds

The bonds involved in the formation of lectin–carbohydrate complexes are in principle not different from those involved in the formation of the corresponding complexes of other carbohydrate-binding proteins. Lectins combine with their ligands primarily by a network of hydrogen bonds and hydrophobic interactions; in rare cases, electrostatic interactions (or ion pairing) and coordination with metal ions also play a role. Bonding is sometimes mediated by one or more water molecules (explained later). Although in a single lectin a limited set of residues contribute to the interactions with the ligand, on the whole almost all kinds of amino acids participate in ligand binding.

3.1 Hydrogen Bonds

Hydrogen bonds that are directional are heavily involved in conferring specificity to protein–carbohydrate interactions, as well as contributing to their affinity. They depend largely on interactions between the hydroxyls of the...