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 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.BWI-1（荞麦胰蛋白酶抑制剂，buckwheat trypsin inhibitor）属于马铃薯蛋白酶抑制剂I家族（potato inhibitor I family）成员，可抑制T细胞急性淋巴细胞白血病（T-acute lymphoblastic leukemia）细胞增殖，并诱导人类实体瘤细胞系发生细胞凋亡（apoptosis）。本研究报道了BWI-1的重组蛋白rBTI（重组荞麦胰蛋白酶抑制剂，recombinant buckwheat trypsin inhibitor）的1.84埃分辨率晶体结构，以及rBTI与牛胰蛋白酶（bovine trypsin）复合物的2.26埃分辨率晶体结构。当rBTI与胰蛋白酶结合后，其P8′位点的色氨酸53（Trp53）发生构象变化，这一现象在既往报道的马铃薯蛋白酶抑制剂I家族其他成员中尚未被观察到。本研究通过测定4种在P2和P8′位点带有不同氨基酸替换的rBTI突变体与胰蛋白酶的结合和解离速率，探究了P8′残基在马铃薯蛋白酶抑制剂I家族中的作用。结果发现，其中一株突变体P44T的抑制活性远高于野生型rBTI，其解离常数达到皮摩尔（pM）级别。本研究结果可为未来设计基于rBTI的新型抗肿瘤药物提供重要参考。