Structural pathway of regulated substrate transfer and threading through an Hsp100 disaggregase.
Structural pathway of regulated substrate transfer and threading through an Hsp100 disaggregase.
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DOI：
10.1126/sciadv.1701726
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发表时间：
2017-08
期刊：
影响因子：
13.6
通讯作者：
Saibil HR
中科院分区：
文献类型：
--
作者：
Deville C;Carroni M;Franke KB;Topf M;Bukau B;Mogk A;Saibil HR
ClpB 复合物的冷冻电镜结构揭示了蛋白质解聚过程中底物转移和易位的途径。重新折叠聚集蛋白对于对抗细胞蛋白毒性应激至关重要。包括大肠杆菌 ClpB 在内的 Hsp100 伴侣与 Hsp70 一起形成强大的解聚机器，将聚集的多肽穿过串联三磷酸腺苷酶 (ATPase) 环的中心孔。为了可视化蛋白质解聚，我们在腺苷 5'-O-(3-硫代三磷酸) 存在下确定了无活性和底物结合 ClpB 的冷冻电子显微镜结构，揭示了具有明显接缝的闭合 AAA+ 环。在无基底状态下，明显的分辨率梯度（可能与迁移率相对应）跨越 AAA+ 环，并在接缝处有动态热点。在接缝侧，卷绕线圈调节域被锁定在水平、非活动方向。另一方面，调节域可用于 Hsp70 结合、底物靶向和激活。在存在模型底物酪蛋白的情况下，多肽穿过整个孔通道，并且核苷酸占用率的增加与更高的 ATP 酶活性相关。底物诱导的结构域位移表明受调节的底物从 Hsp70 转移到 ClpB 孔的途径，在该孔内，螺旋环与底物接触。接缝孔环经历显着的位移，以及调节域的排序。这些不对称运动表明了分解过程中 ATP 酶激活和底物穿线的机制。
Cryo-EM structures of ClpB complexes reveal a pathway for substrate transfer and translocation during protein disaggregation. Refolding aggregated proteins is essential in combating cellular proteotoxic stress. Together with Hsp70, Hsp100 chaperones, including Escherichia coli ClpB, form a powerful disaggregation machine that threads aggregated polypeptides through the central pore of tandem adenosine triphosphatase (ATPase) rings. To visualize protein disaggregation, we determined cryo–electron microscopy structures of inactive and substrate-bound ClpB in the presence of adenosine 5′-O-(3-thiotriphosphate), revealing closed AAA+ rings with a pronounced seam. In the substrate-free state, a marked gradient of resolution, likely corresponding to mobility, spans across the AAA+ rings with a dynamic hotspot at the seam. On the seam side, the coiled-coil regulatory domains are locked in a horizontal, inactive orientation. On the opposite side, the regulatory domains are accessible for Hsp70 binding, substrate targeting, and activation. In the presence of the model substrate casein, the polypeptide threads through the entire pore channel and increased nucleotide occupancy correlates with higher ATPase activity. Substrate-induced domain displacements indicate a pathway of regulated substrate transfer from Hsp70 to the ClpB pore, inside which a spiral of loops contacts the substrate. The seam pore loops undergo marked displacements, along with ordering of the regulatory domains. These asymmetric movements suggest a mechanism for ATPase activation and substrate threading during disaggregation.
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DOI：
10.1016/j.ymeth.2016.03.007
发表时间：
2016-05-01
期刊：
Methods (San Diego, Calif.)
影响因子：
--
作者：
Joseph AP;Malhotra S;Burnley T;Wood C;Clare DK;Winn M;Topf M
通讯作者：
Topf M
影响因子：
4.8
作者：
Lipinska, Natalia;Zietkiewicz, Szymon;Liberek, Krzysztof
通讯作者：
Liberek, Krzysztof
影响因子：
16.8
作者：
Oguchi, Yuki;Kummer, Eva;Bukau, Bernd
通讯作者：
Bukau, Bernd
影响因子：
16
作者：
Kim, YI;Burton, RE;Baker, TA
通讯作者：
Baker, TA
影响因子：
48
作者：
Punjani, Ali;Rubinstein, John L.;Brubaker, Marcus A.
通讯作者：
Brubaker, Marcus A.