Section 23.4.4.2.2. T4 lysozyme

Over 150 mutants of T4 lysozyme have been studied to date, and, for the majority of these, the crystal structures are available. Although most of the mutant structures crystallize isomorphously to the wild type, many of them provide a view of the molecule in different crystal environments. This collection of structures leads to the comparative analysis of the solvent positions in ten different crystal forms of T4 lysozyme, providing a clear picture of the effect of crystal contacts on the hydration sphere of a protein viewed by X-ray crystallography (Zhang & Matthews, 1994). The resolution and degree of refinement of the structures involved varied significantly, from 2.6 to 1.7 Å resolution, and the number of water molecules included per protein molecule ranged from 38 to 160. Nevertheless, this study revealed important features. A striking observation is that 95% of the solvent-exposed residues on T4 lysozyme were involved in at least one crystal contact in one or another of the crystal forms studied, showing that any part of the protein surface can be involved in crystal contacts. A corollary to this finding is that any of the surface water molecules can be displaced or involved in bridging protein–protein contacts in the crystal.

Of the 1675 individual water molecules observed in the 18 independently refined T4 lysozyme molecules included (Fig. 23.4.4.9), the ones that were within a sphere of radius 1.2 Å were considered to occupy the same site on the protein. As in the case of elastase described above, all of the water molecules observed upon superposition of the 18 T4 lysozyme structures represent a large portion of the first hydration shell. This reinforces the concept that multiple structures of a protein of interest provide a more complete picture of the protein hydration than possible with a single structure. There are four buried water sites that are occupied in at least 15 out of the 18 structures and are independent of crystal contacts. Two of these buried sites are at the hinge-bending region between the two helical domains and appear to play a functional role in the opening and closing of the active site (Weaver & Matthews, 1987). The other two play a structural role at the protein core. Other than the four buried water molecules, the most conserved water sites appear at the active-site cleft between the two domains and at the N-termini of α-helices. As is the case in the previous works reviewed above, the 20 most conserved water sites appear in well conserved protein environments and generally have low temperature factors. Buried or highly conserved water molecules also tend to make at least three hydrogen bonds with protein atoms or other water molecules. The less-conserved water sites appear more randomly on the protein surface and are strongly influenced by the particular crystal environment in which the structure was solved.

Figure 23.4.4.9| top | pdf | Distribution of solvent-binding sites in 18 mutant T4 lysozymes from ten refined crystal structures. The lysozyme structures were compared to identify common sites of hydration. A total of 1675 solvent molecules were included in the comparison. Each solvent molecule is represented by a coloured sphere. The size of the sphere is proportional to the number of lysozyme structures in which solvent was observed at the same site (i.e. within 1.2 Å). In addition, the colour of the solvent changes from blue for the least-conserved sites to red for the most-conserved ones [e.g. the red spheres indicate that solvent is observed with high frequency (15–17 times) at the four internal sites]. The numbers indicate representative residue positions along the backbone of the lysozyme molecule. Reprinted with the permission of Cambridge University Press from Zhang & Matthews (1994). Copyright (1994) The Protein Society.