title: Active site binding loop stabilization in the subtilisin inhibitor eglin c: structural and functional studies on specifically designed mutants in complex with subtilisin and the uncomplexed inhibitor.
abstract: As known from the x-ray crystal structure in complex with a proteinase and from NMR studies, the serine proteinase inhibitor eglin c has a wedge-like shape with a hydrophobic core and a solvent exposed active site binding loop which is stabilized by a network of non-covalent core-binding loop interactions. Previous studies implied a crucial role of the P1'-residue Asp-46 for binding loop stabilization and high inhibitory potency of eglin c towards serine proteinases such as subtilisin. In the present study, the formation of specific eglin core-binding loop interactions was modulated by replacing the wildtype Asp-46 by asparagine, glutarnate and glutamine. The x-ray crystal structures of these mutants were solved in complex with subtilisin, and the inhibitory potency towards this enzyme was determined. Our results imply a reduction of inhibitory potency with declining core-binding loop interactions. We succeeded in crystallizing free wildtype eglin c. The 1.95 angstroms x-ray crystal structure indicates that the transition from the free to the bound form of eglin is accompanied by a concerted conformational change in the binding loop, implying an induced fit to the accessible enzyme surface. Except for the binding loop domain and a few residues on the surface of eglin, the differences observed between the uncomplexed and bound form of the inhibitor are only small.
authors.affiliation: Department of Biotechnology, Pharmaceuticals Division, Ciba-Geigy Ltd., Basel, Switzerland.
authors.forename: K
authors.initials: K
authors.lastname: Hipler
authors.forename: J P
authors.initials: JP
authors.lastname: Priestle
authors.forename: J
authors.initials: J
authors.lastname: Rahuel
authors.forename: M G
authors.initials: MG
authors.lastname: Grütter
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray; D004355:Drug Stability; D009154:Mutation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011506:Proteins; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D015843:Serpins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000037:antagonists & inhibitors*
chemical_list: D011506:Proteins; D015842:Serine Proteinase Inhibitors; D015843:Serpins; C028656:eglin proteinase inhibitors; D013381:Subtilisins