Mechanism of the kinetically-controlled folding reaction of subtilisin
Mechanism of the kinetically-controlled folding reaction of subtilisin
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DOI：
10.1021/bi061600z
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发表时间：
2007-01-23
期刊：
影响因子：
2.9
通讯作者：
Bryan, Philip N.
中科院分区：
文献类型：
--
作者：
Fisher, Kathryn E.;Ruan, Biao;Bryan, Philip N.
与许多分泌型蛋白酶一样，枯草杆菌蛋白酶在成熟形式下是动力学稳定的，但在没有其前结构域的帮助下不能折叠。折叠的高动力学障碍的存在挑战了许多被广泛接受的想法，即热力学确定天然结构和热力学稳定性的充分性来确定途径。这篇文章的目的是阐明枯草杆菌蛋白酶折叠的动力学障碍的物理性质，并显示前结构域如何克服这些障碍。为了解决这些问题，我们研究了枯草杆菌蛋白酶前结构域的双分子折叠反应，并设计了一系列枯草杆菌蛋白酶突变体，以探索折叠反应的步骤。我们的分析表明，枯草杆菌蛋白酶的成熟形式的异常缓慢的折叠结果从两个缓慢和连续的过程的累积效应：（1）形成一个不稳定的和拓扑挑战的中间体和（2）脯氨酸限制异构化的中间体的天然状态。新生折叠中间体的低稳定性部分是由于枯草杆菌蛋白酶的稳定性高度依赖于金属结合。天然枯草杆菌蛋白酶在不存在结合金属的情况下是不稳定的。然而，由于这两个金属结合位点是在折叠后期形成的，它们对折叠中间体的稳定性贡献很小。在β链S2和S3之间形成左手交叉连接的拓扑挑战进一步阻碍了生产性折叠中间体的形成。这种连接对于传播折叠反应至关重要。在前结构域的存在下，折叠通过一个主要的中间体进行，该中间体通过前结构域结合而稳定，独立于金属浓度和脯氨酸异构化状态。前结构域还催化形成金属位点B所需的后期脯氨酸异构化。限速脯氨酸异构化在蛋白质折叠中是常见的，但其在减缓枯草杆菌蛋白酶折叠中的作用被放大，因为中间体的不稳定性和明显需要同时异构化多个脯氨酸以产生金属位点B。因此，枯草杆菌蛋白酶的动力学控制的折叠反应，虽然不寻常，是由其他蛋白质中发现的事件的累积效应解释。
Like many secreted proteases, subtilisin is kinetically stable in the mature form but unable to fold without assistance from its prodomain. The existence of high kinetic barriers to folding challenges many widely accepted ideas, namely, the thermodynamic determination of native structure and the sufficiency of thermodynamic stability to determine a pathway. The purpose of this article is to elucidate the physical nature of the kinetic barriers to subtilisin folding and to show how the prodomain overcomes these barriers. To address these questions, we have studied the bimolecular folding reaction of the subtilisin prodomain and a series of subtilisin mutants, which were designed to explore the steps in the folding reaction. Our analysis shows that inordinately slow folding of the mature form of subtilisin results from the accrued effects of two slow and sequential processes: (1) the formation of an unstable and topologically challenged intermediate and (2) the proline-limited isomerization of the intermediate to the native state. The low stability of nascent folding intermediates results in part from subtilisin's high dependence on metal binding for stability. Native subtilisin is thermodynamically unstable in the absence of bound metals. Because the two metal binding sites are formed late in folding, however, they contribute little to the stability of folding intermediates. The formation of productive folding intermediates is further hindered by the topological challenge of forming a left-handed crossover connection between beta-strands S2 and S3. This connection is critical to propagate the folding reaction. In the presence of the prodomain, folding proceeds through one major intermediate, which is stabilized by prodomain binding, independent of metal concentration and proline isomerization state. The prodomain also catalyzes the late proline isomerizations needed to form metal site B. Rate-limiting proline isomerization is common in protein folding, but its effect in slowing subtilisin folding is amplified because of the instability of the intermediate and an apparent need for simultaneous isomerization of multiple prolines in order to create metal site B. Thus, the kinetically controlled folding reaction of subtilisin, although unusual, is explained by the accrued effects of events found in other proteins.