Deciphering the molecular organization of GET pathway chaperones through native mass spectrometry.
Deciphering the molecular organization of GET pathway chaperones through native mass spectrometry.
复制标题
通过天然质谱破译 GET 途径伴侣的分子组织。
DOI：
10.1016/j.bpj.2022.02.026
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发表时间：
2022
影响因子：
3.4
通讯作者：
Gupta,Kallol
中科院分区：
文献类型：
--
作者：
Giska,Fabian;Mariappan,Malaiyalam;Bhattacharyya,Moitrayee;Gupta,Kallol
Get3/4/5伴侣复合体负责将C末端尾部锚定的膜蛋白靶向内质网。尽管存在几种独立蛋白质的晶体结构和亚复合体的部分结构，但对于所涉及的蛋白质复合体，人们提出了不同的寡聚态和结构组织模型。在这里，使用自然质谱学(Native-MS)，结合完整的解离，我们证明了Get4/5通过Get5/5和一个新的Get4/4二聚界面独占地形成了四聚体。在此基础上加入Get3得到具有闭环环状结构的六聚体(Get3)2-(Get4)2-(Get5)2。我们通过分子建模和对所提议的界面的突变废除来进一步验证我们的主张。Native-MS已成为确定蛋白质寡聚组织状态的主要工具。这项工作表明，对于多蛋白质复合体，天然MS结合分子建模和突变扰动，可以提供一种替代途径来呈现低聚状态以及所涉及的分子界面的详细信息。这对于具有大的非结构结构域的大的多蛋白复合体特别有用，这使得它与传统的结构确定方法背道而驰。
Get3/4/5 chaperone complex is responsible for targeting C-terminal tail-anchored membrane proteins to the endoplasmic reticulum. Despite the availability of several crystal structures of independent proteins and partial structures of subcomplexes, different models of oligomeric states and structural organization have been proposed for the protein complexes involved. Here, using native mass spectrometry (Native-MS), coupled with intact dissociation, we show that Get4/5 exclusively forms a tetramer using both Get5/5 and a novel Get4/4 dimerization interface. Addition of Get3 to this leads to a hexameric (Get3)2-(Get4)2-(Get5)2complex with closed-ring cyclic architecture. We further validate our claims through molecular modeling and mutational abrogation of the proposed interfaces. Native-MS has become a principal tool to determine the state of oligomeric organization of proteins. The work demonstrates that for multiprotein complexes, native-MS, coupled with molecular modeling and mutational perturbation, can provide an alternative route to render a detailed view of both the oligomeric states as well as the molecular interfaces involved. This is especially useful for large multiprotein complexes with large unstructured domains that make it recalcitrant to conventional structure determination approaches.