Multi-domain inhibitors capable to block the activity of different classes of proteases are not very common in nature. However, these kinds of molecules are attractive systems for biomedical or biotechnological applications, where two or more different targets need to be neutralized. SmCI, the Sabellastarte magnifica Carboxypeptidase Inhibitor, is a tri-domain BPTI-Kunitz inhibitor capable to inhibit serine proteases and A-like metallocarboxypeptidases. The BPTI-Kunitz family of proteins includes voltage gated channel blockers and inhibitors of serine proteases. SmCI is therefore, the only BPTI-Kunitz protein capable of inhibiting metallocarboxypeptidases. The X-ray structure of the SmCI-carboxypeptidase A complex previously obtained by us, revealed that this enzyme interacts with SmCI N-tail. In the complex, the reactive loops for serine protease inhibition remain fully exposed to the solvent in each domain, suggesting SmCI can simultaneously interact with multiple serine proteases. The twofold goals of this study were: i) to establish serine proteases-SmCI binding stoichiometry, given that the inhibitor is comprised of three potential binding domains; and ii) to determine whether or not SmCI can simultaneously bind both classes of enzymes, to which it binds individually. Our experimental approach included a variety of techniques for the study of protein-protein interactions, using as model enzymes pancreatic trypsin, elastase and carboxypeptidase A. In particular, we combined information obtained from gel filtration chromatography, denaturing electrophoresis, nuclear magnetic resonance spectroscopy and enzyme inhibition assays. Our results show that SmCI is able to bind three trypsin molecules under saturating conditions, but only one elastase interacts with the inhibitor. Additionally, we demonstrated that SmCI can bind serine proteases and carboxypeptidases at the same time (at least in the ratio 1:1:1), becoming the first protease inhibitor that simultaneously block these two mechanistic classes of enzymes.

能够阻断不同类型蛋白酶活性的多结构域抑制剂在自然界中不是很常见。但是，这些分子对于生物医学或生物技术应用来说是有吸引力的系统，其中两个或更多不同的目标需要被中和。SmCI，Sabellastarte壮丽羧肽酶抑制剂是一种三域BPTI-Kunitz抑制剂，能够抑制丝氨酸蛋白酶和A样金属羧肽酶。BPTI-Kunitz蛋白家族包括电压门控通道阻滞剂和丝氨酸蛋白酶抑制剂。因此，SmCI是唯一能够抑制金属羧肽酶的BPTI-Kunitz蛋白。我们先前获得的SmCI-羧肽酶A复合物的X射线结构表明，该酶与SmCI N-tail相互作用。在复合物中，丝氨酸蛋白酶抑制的反应环在每个域中仍完全暴露于溶剂，这表明SmCI可以同时与多种丝氨酸蛋白酶相互作用。这项研究的双重目标是：一）考虑到抑制剂由三个潜在的结合域组成，建立丝氨酸蛋白酶-SmCI结合化学计量；和ii）确定SmCI是否可以同时结合两种酶，使其分别结合。我们的实验方法包括多种研究蛋白质-蛋白质相互作用的技术，使用胰胰蛋白酶，弹性蛋白酶和羧肽酶A作为模型酶。特别是，我们结合了从凝胶过滤色谱，变性电泳，核磁共振光谱和酶获得的信息抑制试验。我们的结果表明，SmCI在饱和条件下能够结合三个胰蛋白酶分子，但是只有一种弹性蛋白酶与抑制剂相互作用。此外，我们证明SmCI可以同时结合丝氨酸蛋白酶和羧肽酶（至少以1：1：1的比例），