Locating interaction sites on proteins: The crystal structure of thermolysin soaked in 2% to 100% isopropanol

Abstract

Multiple-solvent crystal structure determination (MSCS) allows the position and orientation of bound solvent fragments to be identified by determining the structure of protein crystals soaked in organic solvents. We have extended this technique by the determination of high-resolution crystal structures of thermolysin (TLN), generated from crystals soaked in 2% to 100% isopropanol. The procedure causes only minor changes to the conformation of the protein, and an increasing number of isopropanol interaction sites could be identified as the solvent concentration is increased. Isopropanol occupies all four of the main subsites in the active site, although this was only observed at very high concentrations of isopropanol for three of the four subsites. Analysis of the isopropanol positions shows little correlation with interaction energy computed using a molecular mechanics force field, but the experimentally determined positions of isopropanol are consistent with the structures of known protein-ligand complexes of TLN.

DIALS: implementation and evaluation of a new integration package

“…Phasing. Phases were determined by molecular replacement with Phaser (McCoy et al, 2007) using PDB entry 2tlx (English et al, 1999) as the search model with all water molecules and ligands removed. The phasing was straightforward, with a TFZ score of >8, an LLG of >160, a refined LLG of 8684 and one molecule in the asymmetric unit.…”

Computational mapping identifies the binding sites of organic solvents on proteins

“…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.…”