Conformational flexibility of GRASP protein and its constituent PDZ subdomains reveals structural basis of its promiscuous interactome
Conformational flexibility of GRASP protein and its constituent PDZ subdomains reveals structural basis of its promiscuous interactome
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DOI：
10.1101/666495
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发表时间：
2019-06
期刊：
影响因子：
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通讯作者：
L. Mendes;M. Batista;P. Judge;A. Watts;C. Redfield;A. Costa-Filho
中科院分区：
文献类型：
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作者：
L. Mendes;M. Batista;P. Judge;A. Watts;C. Redfield;A. Costa-Filho
高尔基复合体是分泌途径的核心组成部分，负责真核生物的几种关键细胞功能。该复合体由高尔基体基质组成，高尔基体基质包括高尔基体重组和堆积蛋白（GRASPs），其参与脊椎动物中的脑池堆积和侧向连接。GRASPs在其他过程中也有关键作用，具有与几种不同蛋白质结合伴侣相互作用的不寻常能力。GRASP家族的保守N-末端包括两个PDZ结构域。以前的直向同源物的晶体学研究表明，PDZ 1和PDZ 2具有相似的构象和二级结构的内容，但PDZ 1单独介导的GRASP和它们的结合伙伴之间的几乎所有的相互作用。本研究利用核磁共振、同步辐射圆二色性和分子动力学等技术对两种PDZ结构域的结构、柔性和稳定性进行了研究。GRASP PDZ以不寻常的β 3 α 1 β 4 β 5 α 2 β 6 β 1 β 2二级结构排列，NMR数据表明PDZ 1结合口袋由稳定的β 2-链和更灵活且不稳定的α 2-螺旋形成，这表明对较高的PDZ 1混杂性的解释。用分子动力学模拟计算了两个PDZ结构域的构象自由能分布。这些数据表明，结合后，蛋白质伴侣显着减少了构象空间，GRASPs可以通过稳定一个特定的构象，在合作伙伴依赖的方式访问。PDZ 1的结构灵活性，由PDZ 2调节，以及两个PDZ之间的耦合，协调运动，使GRASP与多个合作伙伴相互作用，使它们能够作为混杂的，多任务的蛋白质。高尔基体重组和堆积蛋白（GRASPs）在维持高尔基体结构和非常规蛋白质分泌中起着关键作用。它们广泛的相互作用网络主要由位于蛋白质N端部分的两个PDZ结构域维持。PDZ结构域在相互作用伴侣的数量和多样性方面的不对称性早已被认识到，但这种不对称性的分子决定因素在很大程度上仍然未知。这里提供的生物物理数据为理解为什么PDZ 1与PDZ 2在溶液中的行为不同（尽管它们的3D结构相似）提供了坚实的基础。此外，我们建议，PDZ 2协助配体结合PDZ 1，通过构象稳定。
The Golgi complex is a central component of the secretory pathway, responsible for several critical cellular functions in eukaryotes. The complex is organized by the Golgi matrix, which includes the Golgi Reassembly and Stacking Proteins (GRASPs), which participate in cisternae stacking and lateral linkage in vertebrates. GRASPs also have critical roles in other processes, with an unusual ability to interact with several different protein binding partners. The conserved N-terminus of the GRASP family includes two PDZ domains. Previous crystallographic studies of orthologues suggest that PDZ1 and PDZ2 have similar conformations and secondary structure content, however PDZ1 alone mediates nearly all the interactions between GRASPs and their binding partners. In this work, NMR, Synchrotron-Radiation Circular Dichroism and Molecular Dynamics were used to examine the structure, flexibility and stability of the two constituent PDZ domains. GRASP PDZs are structured in an unusual β3α1β4β5α2β6β1β2 secondary structural arrangement and NMR data indicates that the PDZ1 binding pocket is formed by a stable β2-strand and a more flexible and unstable α2-helix, suggesting an explanation for the higher PDZ1 promiscuity. The conformational free energy profiles of the two PDZ domains were calculated using Molecular Dynamics simulations. The data suggest that, after binding, the protein partner significantly reduces the conformational space that GRASPs can access by stabilizing one particular conformation, in a partner-dependent fashion. The structural flexibility of PDZ1, modulated by PDZ2, and the coupled, coordinated movement between the two PDZs enable GRASPs to interact with multiple partners, allowing them to function as promiscuous, multitasking proteins. Significance Statement Golgi Reassembly and Stacking Proteins (GRASPs) play pivotal roles in the maintenance of Golgi structure as well as in unconventional protein secretion. Their broad network of interactions is mainly sustained by the two-PDZ domains located in the N-terminal portion of the protein. The asymmetry of the PDZ domains in terms of number and diversity of interacting partners has been long recognized, but the molecular determinants of that asymmetry remains largely unknown. The biophysical data presented here provide a firm basis for understanding why PDZ1 behaves differently to PDZ2 in solution, despite their similar 3D structures. Furthermore, we propose that PDZ2 assist ligand binding to PDZ1, by means of conformational stabilization.