Characterizing the tick carboxypeptidase inhibitor - Molecular basis for its two-domain nature
Characterizing the tick carboxypeptidase inhibitor - Molecular basis for its two-domain nature
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DOI：
10.1074/jbc.m602301200
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发表时间：
2006-08-11
影响因子：
4.8
通讯作者：
Calvete, Juan J.
中科院分区：
文献类型：
--
作者：
Arolas, Joan L.;Bronsoms, Silvia;Calvete, Juan J.
壁虱羧肽酶抑制物(TCI)是一种小的、富含二硫键的蛋白质，它选择性地抑制金属羧基肽酶，并强烈促进血栓的纤溶。TCI由两个结构非常相似的结构域组成，每个结构域包含三个以几乎相同的方式排列的二硫键。通过对酸性折叠中间体的动力学和结构分析，表征了TCI及其分离结构域的氧化折叠和还原去折叠途径。TCI折叠通过1-、2-、3-、4-、5-和6-二硫键物种的顺序形成而达到天然形式。TCI的折叠中间体由两个主要的3-二硫键物种(命名为IIIa和IIIb)和一个主要的6-二硫键杂乱异构体(Xa)组成，它们在反应过程中连续积累，并被蛋白质二硫键异构酶的存在强烈阻止。本研究证明了IIIa和IIIb是3-二硫键物种，分别含有TCI的N-末端和C-末端结构域的天然二硫键对，并解释了为什么TCI的两个结构域依次独立折叠。此外，我们还证明了TCI的还原展开经历了两个主要的独立展开事件，即通过形成IIIa和IIIb中间体。综上所述，TCI与分离结构域的折叠、稳定性和抑制活性的比较揭示了该蛋白具有双结构域性质的原因：这两个结构域都有助于其与羧基肽酶的双头结合的特异性和高亲和力。本文的结果为设计更有效和更有选择性的TCI分子提供了有价值的信息。
Tick carboxypeptidase inhibitor (TCI) is a small, disulfide-rich protein that selectively inhibits metallocarboxypeptidases and strongly accelerates the fibrinolysis of blood clots. TCI consists of two domains that are structurally very similar, each containing three disulfide bonds arranged in an almost identical fashion. The oxidative folding and reductive unfolding pathways of TCI and its separated domains have been characterized by kinetic and structural analysis of the acid-trapped folding intermediates. TCI folding proceeds through a sequential formation of 1-, 2-, 3-, 4-, 5-, and 6-disulfide species to reach the native form. Folding intermediates of TCI comprise two predominant 3-disulfide species (named IIIa and IIIb) and a major 6-disulfide scrambled isomer (Xa) that consecutively accumulate along the reaction and are strongly prevented by the presence of protein disulfide isomerase. This study demonstrates that IIIa and IIIb are 3- disulfide species containing the native disulfide pairings of the N- and C-terminal domains of TCI, respectively, and explains why the two domains of TCI fold sequentially and independently. Also, we show that the reductive unfolding of TCI undergoes two main independent unfolding events through the formation of IIIa and IIIb intermediates. Together, the comparison of the folding, stability, and inhibitory activity of TCI with those of the isolated domains reveals the reasons behind the two-domain nature of this protein: both domains contribute to the specificity and high affinity of its double-headed binding to carboxypeptidases. The results obtained herein provide valuable information for the design of more potent and selective TCI molecules.