Cluster analysis of consensus water sites in thrombin and trypsin shows conservation between serine proteases and contributions to ligand specificity

Abstract

Cluster analysis is presented as a technique for analyzing the conservation and chemistry of water sites from independent protein structures, and applied to thrombin, trypsin, and bovine pancreatic trypsin inhibitor (BPTI) to locate shared water sites, as well as those contributing to specificity. When several protein structures are superimposed, complete linkage cluster analysis provides an objective technique for resolving the continuum of overlaps between water sites into a set of maximally dense microclusters of overlapping water molecules, and also avoids reliance on any one structure as a reference. Water sites were clustered for ten superimposed thrombin structures. three trypsin structures, and four BPTI structures. For thrombin, 19% of the 708 microclusters, representing unique water sites, contained water molecules from at least half of the structures, and 4% contained waters from all 10. For trypsin, 77% of the 106 microclusters contained water sites from at least half of the structures, and 57% contained waters from all three. Water site conservation correlated with several environmental features: highly conserved microclusters generally had more protein atom neighbors, were in a more hydrophilic environment, made more hydrogen bonds to the protein, and were less mobile. There were significant overlaps between thrombin and trypsin conserved water sites, which did not localize to their similar active sites, but were concentrated in buried regions including the solvent channel surrounding the Na' site in thrombin, which is associated with ligand selectivity. Cluster analysis also identified water sites conserved in thrombin but not trypsin, and vice versa, providing a list of water sites that may contribute to ligand discrimination. Thus, in addition to facilitating the analysis of water sites from multiple structures, cluster analysis provides a useful tool for distinguishing between conserved features within a protein family and those conferring specificity.

Conserved Water Molecules Stabilize the Ω-Loop in Class A β-Lactamases

“…A total of 13 water molecules were conserved in more than 90% of the structures, including the two water molecules present in all structures. About the same number of conserved waters was found in previous studies of different proteins (5,24,27,33), despite the fact that those studies differ in the method applied, the number of structures, and their resolution. In general, the number of water molecules in crystallographic models increases with resolution (7), while crystallization conditions have only a moderate effect (22).…”

“…All structures including the crystal water molecules were superimposed by multiple-structure fitting of the C ␣ atoms, using the McLachlan algorithm (23) The subsets of atoms to be considered in the fitting process were initially determined by a Needleman-Wunsch sequence alignment and then iteratively updated during the fitting process. Subsequently, a complete linkage hierarchical cluster analysis of the water molecules in all 49 superimposed structures was performed using WatCH software (33). The clustering process groups together water molecules from different structures that physically overlap and represent one specific water position.…”

“…Comparative studies of crystal structures have shown that there are water binding sites on the surface and in the interior of a protein which are occupied by a water molecule in different crystal structures. Cluster analysis and density-based approaches have been used successfully for the identification and analysis of these conserved waters in multiple crystal structures of a protein or in crystal structures of a protein family (4,5,24,27,33).…”

Unexpected crucial role of residue 225 in serine proteases

“…3). The architecture of this channel is highly conserved in serine proteases and is thought to play a key role in substrate recognition and specificity (26)(27)(28). In the Y225F mutant, the side chain of F225 is 90% buried in a hydrophobic pocket located on the outer wall of the primary specificity site and is positioned as Y225 in the wild type (11,24).…”

Electrostatic properties in the catalytic site of papain: A possible regulatory mechanism for the reactivity of the ion pair

“…The justification for this approach consider the fact that in the active site of enzymes the solvent can be affected by strong electric fields and can assume a specific structural ordering. [75][76][77] In this situation, the dielectric response of the active site molecules will not be accurately described in the continuum electrostatic approximation using a high (⑀ s ϭ 80) dielectric constant inside the catalytic cleft.…”