Multiple Solvent Crystal Structures of Chymotrypsin: Comparision with Elastase

Multiple solvent crystal structures of chymotrypsin were used to map the binding surface of the enzyme. Crystals of chymotrypsin grown in aqueous mother liquor were crosslinked with gluteraldehyde and transferred to a variety of organic solvents, whose molecules are representative of functional groups that might appear in a larger ligand. Crystal structures of crosslinked chymotrypsin were solved in 60% acetonitrile, 70% glycerol, 75% isopropanol, 75% t ‐butanol, 50% DMSO, 50%DMF, 75% 1,4‐dioxane, n ‐hexane, 50%cyclopentanol, 50% cyclopentanone, 40% cyclohexanol and 60% cyclohexanone. The chymotrypsin structures in organic solvents are isomorphous with those in aqueous solution. The sites where the solvent molecules bind to the protein were identified directly. All the structures were compared based on a protein backbone least squares superposition. The crystal structures of chymotrypsin in organic solvents showed that the organic solvents clustered in the S1 binding pocket that is occupied by the known inhibitors in chymotrypsin‐inhibitor complexes. Solvent molecules were also found in S1′ and S3′ binding pockets. There was a single dimethylformamide molecule in the oxyanion hole. Unclustered molecules were mostly found in crystal contacts. The pattern of organic solvent molecule clusters determined by MSCS for chymotrypsin is compared with the pattern observed in the multiple solvent crystal structures of a homologous enzyme porcine pancreatic elastase. The MSCS method provides a powerful strategy to map the binding site of proteins helping to determine features that in the future may be helpful in guiding ligand docking and design. This project is funded by National Science Foundation.