Experimental and computational mapping of the binding surface of a crystalline protein

Abstract

Multiple Solvent Crystal Structures (MSCS) is a crystallographic technique to identify energetically favorable positions and orientations of small organic molecules on the surface of proteins. We determined the high-resolution crystal structures of thermolysin (TLN), generated from crystals soaked in 50--70% acetone, 50--80% acetonitrile and 50 mM phenol. The structures of the protein in the aqueous-organic mixtures are essentially the same as the native enzyme and a number of solvent interaction sites were identified. The distribution of probe molecules shows clusters in the main specificity pocket of the active site and a buried subsite. Within the active site, we compared the experimentally determined solvent positions with predictions from two computational functional group mapping techniques, GRID and Multiple Copy Simultaneous Search (MCSS). The experimentally determined small molecule positions are consistent with the structures of known protein--ligand complexes of TLN.

Computational mapping identifies the binding sites of organic solvents on proteins

“…English et al (9,10) determined high resolution crystal structures of TLN, generated from crystals soaked in aqueous solutions of IPA, ACN, CCN, and IPH, and found that the main substrate specificity pocket (S 1 Ј) of the active site binds all four molecules. IPA also binds to the other three subsites of the active site, and additional sites have been observed for IPA, ACN, and IPH as the concentration of organic solvents in the soaking solution increases.…”

“…This site-mapping and fragment-assembly strategy provides an important approach to drug design (1)(2)(3)(4)(5)(6). Experimental approaches to mapping include x-ray crystallography (7)(8)(9)(10) and NMR techniques (11, l2). In the multiple solvent crystal structure method (7)(8)(9)(10), the x-ray structure of a protein is repeatedly solved in a variety of organic solvents, each representing a different functional group.…”

“…Experimental approaches to mapping include x-ray crystallography (7)(8)(9)(10) and NMR techniques (11, l2). In the multiple solvent crystal structure method (7)(8)(9)(10), the x-ray structure of a protein is repeatedly solved in a variety of organic solvents, each representing a different functional group. In NMR methods, the binding of small molecules in aqueous solution is detected by chemical shifts of the protein and by the observation of intermolecular nuclear Overhauser effects (NOEs) between protons of the protein and the ligand (11).…”

“…However, both x-ray crystallography (7)(8)(9)(10) and NMR (11) reveal only a limited number of bound ligand positions, and a pocket of the active site tends to form a consensus site that binds many ligands, irrespective of their sizes and polarities. An NMR study by Liepinsh and Otting (11) shows that methanol, methylene chloride, acetonitrile (CCN), acetone (ACN), DMSO, isopropanol (IPA), t-butanol, and urea all bind to the specificity-determining site (site C) of the hen egg-white lysozyme (HEWL).…”

“…An NMR study by Liepinsh and Otting (11) shows that methanol, methylene chloride, acetonitrile (CCN), acetone (ACN), DMSO, isopropanol (IPA), t-butanol, and urea all bind to the specificity-determining site (site C) of the hen egg-white lysozyme (HEWL). Recent multiple solvent crystal structure studies of thermolysin (TLN) (9,10) show that IPA, ACN, CCN, and phenol (IPH) bind preferentially to subsite S 1 Ј of the active site.…”