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首页> 外文期刊>Journal of Molecular Biology >Structural studies on the co-chaperone Hop and its complexes with Hsp90.
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Structural studies on the co-chaperone Hop and its complexes with Hsp90.

机译：对伴侣伴侣啤酒花及其与Hsp90的复合物的结构研究。

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The tetratricopeptide repeat domain (TPR)-containing co-chaperone Hsp-organising protein (Hop) plays a critical role in mediating interactions between Heat Shock Protein (Hsp)70 and Hsp90 as part of the cellular assembly machine. It also modulates the ATPase activity of both Hsp70 and Hsp90, thus facilitating client protein transfer between the two. Despite structural work on the individual domains of Hop, no structure for the full-length protein exists, nor is it clear exactly how Hop interacts with Hsp90, although it is known that its primary binding site is the C-terminal MEEVD motif. Here, we have undertaken a biophysical analysis of the structure and binding of Hop to Hsp90 using a variety of truncation mutants of both Hop and Hsp90, in addition to mutants of Hsp90 that are thought to modulate the conformation, in particular the N-terminal dimerisation of the chaperone. The results establish that whilst the primary binding site of Hop is the C-terminal MEEVD peptide of Hsp90, binding also occurs at additional sites in the C-terminal and middle domain. In contrast, we show that another TPR-containing co-chaperone, CyP40, binds solely to the C-terminus of Hsp90. Truncation mutants of Hop were generated and used to investigate the dimerisation interface of the protein. In good agreement with recently published data, we find that the TPR2a domain that contains the Hsp90-binding site is also the primary site for dimerisation. However, our results suggest that residues within the TPR2b may play a role. Together, these data along with shape reconstruction analysis from small-angle X-ray scattering measurements are used to generate a solution structure for full-length Hop, which we show has an overall butterfly-like quaternary structure. Studies on the nucleotide dependence of Hop binding to Hsp90 establish that Hop binds to the nucleotide-free, 'open' state of Hsp90. However, the Hsp90-Hop complex is weakened by the conformational changes that occur in Hsp90 upon ATP binding. Together, the data are used to propose a detailed model of how Hop may help present the client protein to Hsp90 by aligning the bound client on Hsp70 with the middle domain of Hsp90. It is likely that Hop binds to both monomers of Hsp90 in the form of a clamp, interacting with residues in the middle domain of Hsp90, thus preventing ATP hydrolysis, possibly by the prevention of association of N-terminal and middle domains in individual Hsp90 monomers.
机译：含有四肽重复结构域（TPR）的伴侣蛋白Hsp组织蛋白（Hop）在介导热激蛋白（Hsp）70和Hsp90之间的相互作用中起着关键作用，而Hsp90是细胞装配机器的​​一部分。它还调节Hsp70和Hsp90的ATPase活性，从而促进两者之间客户蛋白质的转移。尽管在Hop的各个结构域上进行了结构研究，但不存在全长蛋白的结构，也不清楚确切的Hop如何与Hsp90相互作用，尽管已知其主要结合位点是C端MEEVD基序。在这里，我们使用Hop和Hsp90的各种截短突变体，除了认为可以调节构象的Hsp90突变体，特别是N端二聚化，还对Hop的结构和结合进行了生物物理分析。的伴侣。结果证实，虽然Hop的主要结合位点是Hsp90的C端MEEVD肽，但结合也发生在C端和中间结构域的其他位点。相比之下，我们表明另一个包含TPR的伴侣分子CyP40仅与Hsp90的C末端结合。产生了Hop的截短突变体，并用于研究蛋白质的二聚化界面。与最近发表的数据非常吻合，我们发现包含Hsp90结合位点的TPR2a域也是二聚化的主要位点。但是，我们的结果表明TPR2b中的残基可能起作用。这些数据连同来自小角度X射线散射测量的形状重构分析一起用于生成全长Hop的解决方案结构，我们展示了它具有整体蝶形四元结构。关于Hop与Hsp90结合的核苷酸依赖性的研究表明，Hop与Hsp90的无核苷酸，“开放”状态结合。但是，Hsp90-Hop复合物被ATP结合后在Hsp90中发生的构象变化所削弱。总之，这些数据用于提出详细的模型，说明如何通过将Hsp70上的结合的客户与Hsp90的中间结构域对齐来使Hop能够将客户蛋白呈现给Hsp90。 Hop可能以钳位的形式结合到Hsp90的两个单体上，与Hsp90的中间结构域中的残基相互作用，从而防止ATP水解，可能是通过防止单个Hsp90单体中的N末端和中间结构域缔合。

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