Structural investigation of a chaperonin in action reveals how nucleotide binding regulates the functional cycle.
Structural investigation of a chaperonin in action reveals how nucleotide binding regulates the functional cycle.
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DOI：
10.1126/sciadv.aau4196
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发表时间：
2018-09
期刊：
影响因子：
13.6
通讯作者：
Boisbouvier J
中科院分区：
文献类型：
--
作者：
Mas G;Guan JY;Crublet E;Debled EC;Moriscot C;Gans P;Schoehn G;Macek P;Schanda P;Boisbouvier J
位点选择性同位素标记可以通过 NMR 光谱对工作 1-MDa 伴侣蛋白进行结构和功能研究。伴侣蛋白是细菌、真核生物和古细菌中普遍存在的蛋白质组装体，促进蛋白质折叠，防止蛋白质聚集，从而参与维持细胞内的蛋白质稳态。在其功能周期中，它们将未折叠的客户蛋白结合在其双环结构内，并通过以腺苷 5'-三磷酸 (ATP) 依赖性方式关闭环室来促进蛋白质折叠。尽管完全开放和封闭形式的伴侣蛋白的静态结构已通过 X 射线晶体学或电子显微镜解析，但阐明此类 ATP 驱动的分子事件的机制需要在工作条件下在结构水平上研究蛋白质。我们引入了一种方法，它将通过先进的同位素标记方法实现的非常大的蛋白质的位点特异性核磁共振观察与原位 ATP 再生系统相结合。使用这种方法，我们在处理客户蛋白时提供了对 1-MDa 大 hsp60 伴侣蛋白的功能深入了解，并揭示了核苷酸结合、水解和释放控制如何在关闭和开放状态之间切换。虽然开放构象稳定了客户蛋白的未折叠状态，但客户蛋白在伴侣蛋白腔内的内化加速了其功能周期。这种方法为研究各种 ATP 驱动的生物机器在热作用下的结构和机制开辟了新的视角。
Site-selective isotope labeling enables structural and functional investigation of a working 1-MDa chaperonin by NMR spectroscopy. Chaperonins are ubiquitous protein assemblies present in bacteria, eukaryota, and archaea, facilitating the folding of proteins, preventing protein aggregation, and thus participating in maintaining protein homeostasis in the cell. During their functional cycle, they bind unfolded client proteins inside their double ring structure and promote protein folding by closing the ring chamber in an adenosine 5′-triphosphate (ATP)–dependent manner. Although the static structures of fully open and closed forms of chaperonins were solved by x-ray crystallography or electron microscopy, elucidating the mechanisms of such ATP-driven molecular events requires studying the proteins at the structural level under working conditions. We introduce an approach that combines site-specific nuclear magnetic resonance observation of very large proteins, enabled by advanced isotope labeling methods, with an in situ ATP regeneration system. Using this method, we provide functional insight into the 1-MDa large hsp60 chaperonin while processing client proteins and reveal how nucleotide binding, hydrolysis, and release control switching between closed and open states. While the open conformation stabilizes the unfolded state of client proteins, the internalization of the client protein inside the chaperonin cavity speeds up its functional cycle. This approach opens new perspectives to study structures and mechanisms of various ATP-driven biological machineries in the heat of action.
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影响因子：
16.8
作者：
Munoz, Ines G.;Yebenes, Hugo;Montoya, Guillermo
通讯作者：
Montoya, Guillermo
影响因子：
11.4
作者：
Llorca, O;Martín-Benito, J;Valpuesta, JM
通讯作者：
Valpuesta, JM
影响因子：
4.8
作者：
Iizuka, R;Yoshida, T;Yohda, M
通讯作者：
Yohda, M
DOI：
10.1016/j.str.2010.07.009
发表时间：
2010-10-13
期刊：
Structure (London, England : 1993)
影响因子：
--
作者：
Huo Y;Hu Z;Zhang K;Wang L;Zhai Y;Zhou Q;Lander G;Zhu J;He Y;Pang X;Xu W;Bartlam M;Dong Z;Sun F
通讯作者：
Sun F
影响因子：
15
作者：
Amero, Carlos;Schanda, Paul;Boisbouvier, Jerome
通讯作者：
Boisbouvier, Jerome