Section 23.4.3.4. Water mediation of protein–ligand interactions

A series of three papers presents the results of an analysis of water molecules mediating protein–ligand interactions in 19 crystal structures solved to better than 2.0 Å resolution and refined to an R factor of at least 23% (Poormina & Dean, 1995a,b,c). The studies focus on hydrogen-bonding features of water molecules bridging protein–ligand complexes (Poormina & Dean, 1995b), on the surface shape of the protein and ligand molecules at the water-binding sites (Poormina & Dean, 1995c), and on the structural and functional importance of water molecules conserved at the binding sites in five sets of evolutionarily related proteins (Poormina & Dean, 1995a). This study was largely motivated by an attempt to distinguish between properties of water-binding sites where water molecules are displaced by ligands and those where water molecules must be considered as part of the protein surface. This type of understanding has direct implications for drug and ligand design.

In general, there is a strong correlation between the number of water molecules found to bridge any given protein–ligand complex and the number of hydrophilic groups associated with the ligand. Within this context and in agreement with the conclusions of Kuhn et al. (1992), the authors found that the protein shape is important in determining the location of water-binding sites at the protein–ligand interface. Fig. 23.4.3.5 illustrates the different types of grooves observed in this study. Figs. 23.4.3.5(a) and (b) represent binding of bridging water molecules in deep grooves on the protein or on the ligand, respectively. The most common situation is illustrated in Fig. 23.4.3.5(a), with that in Fig. 23.4.3.5(b) occurring very rarely. Fig. 23.4.3.5(c) shows the situation where water molecules are found to interact with the ligand alone or at the periphery of the protein–ligand interface. Finally, Fig. 23.4.3.5(d) illustrates the situation where clusters of water molecules occupy elongated grooves, mediating the protein–ligand interaction. A striking example of this is given by the complex between chloramphenicol acetyl transferase and chloramphenicol, where two clusters of water molecules are found to form a layer between the enzyme and the ligand (Poormina & Dean, 1995c).

Figure 23.4.3.5| top | pdf | Schematic illustration of water molecules bound in different types of grooves between protein and ligand. The hatched surfaces represent the ligand surface. (a) Water molecules bound in an indentation on the protein surface, where the protein surface area exposed to the water molecules is far larger than the ligand surface area; (b) water molecules bound in indentations on the ligand surface, where the ligand surface area exposed to the water molecule is larger than the protein surface area; (c) water molecules bound in shallow grooves at the protein–ligand interface and on the ligand surface; and (d) water molecules bound in clusters in elongated grooves with micro-grooves. Reprinted with permission from Poormina & Dean (1995c). Copyright (1995) Kluwer Academic Publishers.

For the purposes of analysis, the authors distinguish between water molecules that interact with both protein and ligand, forming a bridge between the two, and water molecules that interact with either the protein or the ligand, but not with both. There is also a group of water molecules that interact with neither protein nor ligand, but are thought to contribute to the stability of the network of water molecules at the protein–ligand interface.

Of the 58 water molecules found to bridge between protein and ligand, 38 (nearly 80%) make three or more hydrogen bonds and satisfy tetrahedral geometry. Furthermore, they bind in deep grooves, generally interacting more strongly with the protein (Fig. 23.4.3.5a). The B factors of these bridging water molecules are comparable to those of the protein atoms with which they interact. They can, in effect, be considered an integral part of the protein structure and binding site. Many of these bridging water molecules are conserved throughout homologous proteins, even when different ligands are considered, and are clearly structurally significant in maintaining the properties of the protein binding sites.

Water molecules found to bind in shallow grooves do so either at the ligand surface or at the periphery of the protein–ligand interface. For many of these water molecules, the surface areas of the protein and the ligand exposed to the same water molecule are nearly equal. Water molecules binding in shallow grooves are found to have zero to two polar contacts with the protein and are not particularly well conserved within families of homologous proteins.

In general, the authors conclude that water molecules that are to be considered as part of the protein binding site during the design of a new ligand are those that bind in deep grooves, making multiple hydrogen bonds to protein atoms. These water molecules tend to be conserved through families of homologous proteins. The amino-acid residues that interact with deep-groove water molecules tend to be more conserved compared with other residues interacting with the ligand. Conversely, the binding of water in shallow grooves does not seem to be influenced by any special general feature of the protein or ligand surface, and it would be difficult to select water molecules a priori for inclusion as part of the protein structure during the process of ligand design.