Binding properties of the quaternary assembly protein SPAG1
Binding properties of the quaternary assembly protein SPAG1
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DOI：
10.1042/bcj20190198
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发表时间：
2019-06-14
影响因子：
4.1
通讯作者：
Quinternet, Marc
中科院分区：
文献类型：
--
作者：
Chagot, Marie-Eve;Morais, Raphael Dos Santos;Quinternet, Marc
在细胞中，许多成分能够组装形成具有非常特定的生物和生理功能的大分子机器，例如核糖体、剪接体和蛋白酶体。此类实体的组装通常由瞬时蛋白质因子介导。 SPAG1 是一种多结构域蛋白，已知参与内动力蛋白臂和外动力蛋白臂的组装。这些臂是敏感细胞和运动细胞发挥功能所必需的。 SPAG1 与 RUVBL1、RUVBL2 和 PIH1D2 一起是 R2SP 的关键元件，R2SP 是一种蛋白质复合物，以组织特异性方式协助特定蛋白质客户的四级组装，并与热休克蛋白 (HSP) 和调节因子相关联。在本研究中，我们通过结合生化测定、ITC、NMR 光谱和分子动力学 (MD) 模拟，研究了 SPAG1 的 TPR 结构域在 HSP 伴侣招募中的作用。首先，我们提出三个 TPR 结构域中只有两个能够招募蛋白伴侣 HSP70 和 HSP90。然后，我们重点关注其中一个 TPR 结构域，并使用 NMR 光谱阐明了其 3D 结构。依靠 NMR 驱动的对接方法和 MD 模拟，我们破译了其与 HSP70 和 HSP90 的 C 端尾部的结合界面。最后，我们讨论了SPAG1的生物学功能，并具体证明了SPAG1亚片段（包含假定的P环基序）不能在体外有效结合和水解GTP。我们的数据对 SPAG1 具有 GTPase 活性的解释提出了挑战。相反，我们建议 SPAG1 调节 HSP 和 RUVBL1/2 伴侣的核苷酸水解活性。
In cells, many constituents are able to assemble resulting in large macromolecular machineries possessing very specific biological and physiological functions, e.g. ribosome, spliceosome and proteasome. Assembly of such entities is commonly mediated by transient protein factors. SPAG1 is a multidomain protein, known to participate in the assembly of both the inner and outer dynein arms. These arms are required for the function of sensitive and motile cells. Together with RUVBL1, RUVBL2 and PIH1D2, SPAG1 is a key element of R2SP, a protein complex assisting the quaternary assembly of specific protein clients in a tissue-specific manner and associating with heat shock proteins (HSPs) and regulators. In this study, we have investigated the role of TPR domains of SPAG1 in the recruitment of HSP chaperones by combining biochemical assays, ITC, NMR spectroscopy and molecular dynamics (MD) simulations. First, we propose that only two, out of the three TPR domains, are able to recruit the protein chaperones HSP70 and HSP90. We then focused on one of these TPR domains and elucidated its 3D structure using NMR spectroscopy. Relying on an NMR-driven docking approach and MD simulations, we deciphered its binding interface with the C-terminal tails of both HSP70 and HSP90. Finally, we addressed the biological function of SPAG1 and specifically demonstrated that a SPAG1 sub-fragment, containing a putative P-loop motif, cannot efficiently bind and hydrolyze GTP in vitro. Our data challenge the interpretation of SPAG1 possessing GTPase activity. We propose instead that SPAG1 regulates nucleotide hydrolysis activity of the HSP and RUVBL1/2 partners.