Structural Insights into the Role of the Cyclic Backbone in a Squash Trypsin Inhibitor

Norelle L Daly,Louise Thorstholm,Kathryn P Greenwood,Gordon J King,K Johan Rosengren,Begoña Heras,Jennifer L Martin,David J Craik

doi:10.1074/jbc.m113.528240

Abstract

MCoTI-II is a head-to-tail cyclic peptide with potent trypsin inhibitory activity and, on the basis of its exceptional proteolytic stability, is a valuable template for the design of novel drug leads. Insights into inhibitor dynamics and interactions with biological targets are critical for drug design studies, particularly for protease targets. Here, we show that the cyclization and active site loops of MCoTI-II are flexible in solution, but when bound to trypsin, the active site loop converges to a single well defined conformation. This finding of reduced flexibility on binding is in contrast to a recent study on the homologous peptide MCoTI-I, which suggested that regions of the peptide are more flexible upon binding to trypsin. We provide a possible explanation for this discrepancy based on degradation of the complex over time. Our study also unexpectedly shows that the cyclization loop, not present in acyclic homologues, facilitates potent trypsin inhibitory activity by engaging in direct binding interactions with trypsin.

Highlights

MCoTI-II is a potent serine protease inhibitor with a cyclic protein backbone
Our study reveals a number of interactions between the cyclization loop and trypsin and Extraction of MCoTI-II from M. cochinchinensis—Native MCoTI-II was purified from the seeds of M. cochinchinensis as described previously [19]
Analysis of Backbone Dynamics of MCoTI-II—To determine whether the disorder in the three-dimensional structure of MCoTI-II is a result of flexibility or a lack of distance restraints in the structure calculations, the backbone dynamics of MCoTI-II in solution were analyzed using NMR relaxation experiments

Summary

Background

MCoTI-II is a potent serine protease inhibitor with a cyclic protein backbone. Results: Its three-dimensional structure and dynamics in complex with trypsin were determined using x-ray and NMR methods. An analysis of the dynamics of MCoTI-I bound to trypsin was carried out, and it was reported that several residues, including the active site lysine residue, become more flexible upon binding to trypsin [20] This is a very surprising result given that the MCoTI peptides have picomolar Ki values against trypsin [21] and that protein binding is generally thought to result in reduced rather than increased flexibility [22, 23]. We have analyzed the dynamics of MCoTI-II in solution and determined its crystal structure bound to trypsin. This is the first structure of a peptide containing a cyclic cystine knot motif bound to trypsin and, the first resolution of the structure of any cyclotide bound to a defined macromolecular target.

RESULTS

DISCUSSION

Data collection and refinement statistics