Conformational changes of rBTI from buckwheat upon binding to trypsin: implications for the role of the P(8)' residue in the potato inhibitor I family.

Longfei Wang,Mei Li,Xiaowei Pan,Wenrui Chang,Hongmei Zhang,Yu Gao,Zhuanhua Wang,Peng Cao,Fei Zhao

doi:10.1371/journal.pone.0020950

Abstract

BWI-1 (buckwheat trypsin inhibitor), a member of the potato inhibitor I family, suppresses the growth of T-acute lymphoblastic leukemia cells and induces apoptosis in human solid tumor cell lines. Here, we report the crystal structure of rBTI (recombinant buckwheat trypsin inhibitor), a recombinant protein of BWI-1, at 1.84 Å resolution and the structure of rBTI in complex with bovine trypsin at 2.26 Å resolution. A conformational change of Trp53 at the P8′ position in rBTI was observed upon its binding to trypsin, which is not seen in other members of the potato inhibitor I family reported previously. The role of the P8′ residue in the potato inhibitor I family was examined by measuring the association and dissociation rates of four rBTI mutants with different substitutions at the P2 and P8′ positions when binding to trypsin. One of the mutants, P44T, was found to be a much stronger inhibitor than wild-type rBTI, with a picomolar (pM) dissociation constant. Our results could provide valuable insights for designing a new rBTI-based antitumor drug in the future.

Highlights

Canonical inhibitors of serine protease function according to the standard mechanism of protease inhibition in which they bind tightly in the active site of a cognate protease in a substrate-like manner
Cierpicki et al proposed that because that rBTI presents an uncommon residues (Trp) has a shorter side-chain than Arg, the binding loop of LUTI would be much closer to the protein core than other members of the potato inhibitor I family [14]
Binding loop conformations of the potato inhibitor I family and their inhibition activities Following the discovery of the rotameric switch of the P89 Trp in rBTI, two questions arose: why does rBTI exhibit such a rare inhibition mechanism; and what roles do the P89 and P2 residues play in its inhibition activity? To address these questions, we referred to the structure of several classical members of the potato inhibitor I family and designed four rBTI mutants

Summary

Introduction

Canonical inhibitors of serine protease function according to the standard mechanism of protease inhibition in which they bind tightly in the active site of a cognate protease in a substrate-like manner Several rBTI mutants were constructed to mimic different binding loop conformations of potato inhibitor I family members.

Results

Conclusion