Although digestive resistance of Kunitz protease inhibitors has been reported extensively, the molecular mechanism is not well established. In the present study, the first X-ray structure of Cassia obtusifolia trypsin inhibitor (COTI), a member of Kunitz protease inhibitors, was solved at a resolution of 1.9 Å. The structure adopted a classic β-trefoil fold with the inhibitory loop protruding from the hydrophobic core. The role of Phe139, a unique residue in Kunitz protease inhibitors, and Arg63 in the COTI structure was verified by F139A and R63E mutants. COTI was a specific inhibitor of bovine trypsin and the result was also verified by COTI-trypsin complex formation. Meanwhile, COTI showed equivalent inhibitory activity with that of soybean trypsin inhibitor against bovine trypsin and midgut trypsin from Pieris rapae. The F139 and R63E mutants further indicated that inhibitory specificity and efficiency of COTI were closely related to the global framework, the conformation and the amino acid composition of reactive loop. Finally, a midgut trypsin from P. rapae (PrSP40), which might be involve in the food digestion, was proposed to be a potential target of COTI and might be a promising target for future crop-protection strategy. The results supported the digestive resistance of COTI.

中文翻译：

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决明子胰蛋白酶抑制剂的结构和功能关系，以了解其对菜青虫的消化抗性。

尽管已经广泛报道了Kunitz蛋白酶抑制剂的消化抗性，但是其分子机理尚不明确。在本研究中，解决了决明子胰蛋白酶抑制剂（COTI）（Kunitz蛋白酶抑制剂的一个成员）的第一个X射线结构，分辨率为1.9。该结构采用经典的β-三叶折叠，抑制环从疏水核心突出。F139A和R63E突变体证实了Phe139（Kunitz蛋白酶抑制剂中的独特残基）和Arg63在COTI结构中的作用。COTI是牛胰蛋白酶的特异性抑制剂，其结果也通过COTI-胰蛋白酶复合物的形成得到了验证。同时，COTI显示出与大豆胰蛋白酶抑制剂对菜青虫的牛胰蛋白酶和中肠胰蛋白酶相同的抑制活性。F139和R63E突变体进一步表明COTI的抑制特异性和效率与整体框架，反应性环的构象和氨基酸组成密切相关。最后，有人提出将可能与食物消化有关的菜豆中肠胰蛋白酶（PrSP40）作为COTI的潜在靶标，并且可能是未来作物保护策略的有希望的靶标。结果支持了COTI的消化抗性。建议将其作为COTI的潜在目标，并且可能是未来作物保护策略的有希望的目标。结果支持了COTI的消化抗性。建议将其作为COTI的潜在目标，并且可能是未来作物保护策略的有希望的目标。结果支持了COTI的消化抗性。