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

Summary

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 Pl’-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, glutamate 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 å 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, differences observed between the uncomplexed and bound form of the inhibitor are only small.