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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.

K Hipler·J P Priestle·J Rahuel·M G Grütter
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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.

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Binding SitesCrystallography, X-RayDrug StabilityMutationProtein EngineeringProtein Structure, SecondaryProteinsSerine Proteinase InhibitorsSerpinsStructure-Activity RelationshipSubtilisinsJournal Article

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K Hipler, J P Priestle, J Rahuel, M G Grütter. 1996. 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.. https://doi.org/10.1007/978-1-4613-0319-0_6

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