Cellular folding pathway of a metastable serpin
Cellular folding pathway of a metastable serpin
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DOI：
10.1073/pnas.1603386113
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发表时间：
2016-06-07
影响因子：
11.1
通讯作者：
Hebert, Daniel N.
中科院分区：
文献类型：
--
作者：
Chandrasekhar, Kshama;Ke, Haiping;Hebert, Daniel N.
虽然蛋白质通常折叠到它们的化学上最稳定的状态，但一些亚稳态蛋白质占据更高的自由能状态。从亚稳的较高自由能状态到具有更大稳定性的较低自由能状态的构象变化然后可以产生执行生理学上重要的功能所需的功。然而，亚稳态蛋白质如何在细胞中折叠到这些更高的自由能状态并避免更稳定但无活性的构象却知之甚少。亚稳态蛋白酶抑制剂的丝氨酸蛋白酶抑制剂家族使用自由能下降的大构象变化，通过拉开蛋白酶活性位点来抑制靶蛋白酶。丝氨酸蛋白酶抑制剂抗凝血酶III（ATIII）靶向凝血酶和参与血液凝固的其他蛋白酶，并且ATIII错误折叠可因此导致血栓形成和其他疾病。ATIII具有三个二硫键，两个靠近N末端，一个靠近C末端。我们对ATIII细胞内折叠的研究揭示了二硫键形成的令人惊讶的、有偏见的顺序，在N末端二硫键形成之前，C末端二硫键较早形成，这对于折叠到活性亚稳态至关重要。在这种双结构域蛋白中，主要是β折叠ATIII结构域的早期折叠限制了反应中心环（RCL），该环含有蛋白酶结合位点，确保RCL保持可访问性。N-连接聚糖和碳水化合物结合分子伴侣有助于功能性ATIII的有效折叠和分泌。许多疾病相关的ATIII变体无法导航折叠反应有助于解释其疾病表型。
Although proteins generally fold to their thermodynamically most stable state, some metastable proteins populate higher free energy states. Conformational changes from metastable higher free energy states to lower free energy states with greater stability can then generate the work required to perform physiologically important functions. However, how metastable proteins fold to these higher free energy states in the cell and avoid more stable but inactive conformations is poorly understood. The serpin family of metastable protease inhibitors uses large conformational changes that are downhill in free energy to inhibit target proteases by pulling apart the protease active site. The serpin antithrombin III (ATIII) targets thrombin and other proteases involved in blood coagulation, and ATIII misfolding can thus lead to thrombosis and other diseases. ATIII has three disulfide bonds, two near the N terminus and one near the C terminus. Our studies of ATIII in-cell folding reveal a surprising, biased order of disulfide bond formation, with early formation of the C-terminal disulfide, before formation of the N-terminal disulfides, critical for folding to the active, metastable state. Early folding of the predominantly beta-sheet ATIII domain in this two-domain protein constrains the reactive center loop (RCL), which contains the protease-binding site, ensuring that the RCL remains accessible. N-linked glycans and carbohydrate-binding molecular chaperones contribute to the efficient folding and secretion of functional ATIII. The inability of a number of disease-associated ATIII variants to navigate the folding reaction helps to explain their disease phenotypes.