Mesotrypsin Has Evolved Four Unique Residues to Cleave Trypsin Inhibitors as Substrates
Mesotrypsin Has Evolved Four Unique Residues to Cleave Trypsin Inhibitors as Substrates
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DOI：
10.1074/jbc.m115.662429
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发表时间：
2015-08-28
影响因子：
4.8
通讯作者：
Radisky, Evette S.
中科院分区：
文献类型：
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作者：
Alloy, Alexandre P.;Kayode, Olumide;Radisky, Evette S.
背景：经典丝氨酸蛋白酶抑制剂通常表现为不可裂解的底物;中胰蛋白酶靶向这些抑制剂作为底物。结果：四个空间上分离的氨基酸残基合作，以促进抑制剂切割中胰蛋白酶。结论：抑制剂裂解是一种复杂的进化适应。重要性：人中胰蛋白酶与其他哺乳动物胰蛋白酶具有高度同源性，但其功能独特，对经典丝氨酸蛋白酶抑制剂的抑制具有抗性，并能优先切割这些抑制剂。Arg-193和Ser-39已被鉴定为中胰蛋白酶的抑制剂抗性和切割能力的贡献者，但尚不清楚这些残基是否完全解释了中胰蛋白酶的不寻常性质。在这里，我们使用人阳离子胰蛋白酶作为模板，工程增益的催化功能，评估突变体含有中胰蛋白酶样突变的抗牛胰蛋白酶抑制剂（BPTI）和淀粉样前体蛋白Kunitz蛋白酶抑制剂（APPI）的抑制，并水解这些抑制剂作为底物的能力。我们发现Arg-193和Ser-39足以赋予中胰蛋白酶样抑制抗性;然而，与中胰蛋白酶相比，胰蛋白酶-Y39 S/G193 R双突变体在水解BPTI时仍然慢10倍，在水解APPI时慢2.5倍。我们确定了中胰蛋白酶中的两个额外的残基，Lys-74和Asp-97，其与Arg-193和Ser-39一致，赋予中胰蛋白酶对BPTI和APPI的蛋白水解的完全催化能力。胰蛋白酶突变体与BPTI复合的新晶体结构表明，这四个残基的功能合作，有利于构象动力学，有助于裂解抑制剂的解离。我们的研究结果表明，有效的抑制剂裂解是一个复杂的能力，至少有四个空间上分离的残基中胰蛋白酶的贡献。这些研究结果表明，抑制剂裂解代表了中胰蛋白酶的功能适应，可能已经演变为积极的选择压力。
Background: Canonical serine protease inhibitors normally behave as uncleavable substrates; mesotrypsin targets these inhibitors as substrates. Results: Four spatially separated amino acid residues cooperate to facilitate inhibitor cleavage by mesotrypsin. Conclusion: Inhibitor cleavage is a complex evolutionary adaptation. Significance: Mesotrypsin may regulate a network of serine proteases through its ability to cleave and inactivate multiple protease inhibitors.Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursor protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. These findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.