Conformational Changes of rBTI from Buckwheat upon Binding to Trypsin: Implications for the Role of the P8′ Residue in the Potato Inhibitor I Family

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 P 8 ′ 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 P 8 ′ 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 P 2 and P 8 ′ 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.