Evaluation of chemical cross-linkers for in-depth structural analysis of G protein-coupled receptors through cross-linking mass spectrometry
Evaluation of chemical cross-linkers for in-depth structural analysis of G protein-coupled receptors through cross-linking mass spectrometry
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通过交联质谱法评估化学交联剂对 G 蛋白偶联受体进行深入结构分析
DOI：
10.1016/j.aca.2019.12.036
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发表时间：
2020
影响因子：
6.2
通讯作者：
Wenqing Shui
中科院分区：
文献类型：
--
作者：
Lisha Xia;Ziliang Ma;Jiahui Tong;YuliangTang;Shanshan Li;Shanshan Qin;Ronghui Lou;Suwen Zhao;XiaoguangLei;Wenqing Shui
可以想象，化学交联剂会导致蛋白质的结构破坏，但很少有人在这方面对交联剂进行充分的评估。此外，完整的膜蛋白在选择合适的交联剂方面可能与可溶性蛋白不同，这一点从未被研究过。在这项研究中，我们系统地评估了五种针对Lys、Asp和Glu的传统交联剂和两种Arg反应性交联剂对两个G蛋白偶联受体(GPCRs)结构和功能完整性的影响。观察到受体结构和配体结合活性的扰动，这取决于受体和交联物的条件。特别是，我们的研究表明，PDH和Kargo的浓度需要微调，以最大限度地减少特定GPCRs的结构和功能干扰。选择了一组顺从的交联剂，以获得两个GPCR的最全面的交联图。我们的深度交联质谱(CXMS)分析揭示了GPCRs中晶体结构中看不到的结构区域的动态特征。因此，使用扩展的工具包对GPCRs进行CXMS分析将有助于对未表征的受体进行结构建模，并获得对受体-配体相互作用的新见解。
Chemical cross-linking would conceivably cause structural disruption of a protein, but few cross-linkers have been fully evaluated in this aspect. Furthermore, integral membrane proteins may differ from soluble proteins in the selection of suitable cross-linkers, which has never been investigated. In this study, we systematically evaluated the impact of five conventional cross-linkers targeting Lys, Asp and Glu, and two Arg-reactive cross-linkers on the structural and functional integrity of two G protein-coupled receptors (GPCRs). Perturbation of the receptor structure and ligand-binding activity was observed, depending on the receptor and cross-linking conditions. In particular, our study demonstrated that the concentrations of PDH and KArGO need to be fine-tuned in order to minimize the structural and functional disturbance of specific GPCRs. A set of amenable cross-linkers was selected to acquire the most comprehensive cross-link maps for two GPCRs. Our in-depth cross-linking mass spectrometry (CXMS) analysis has revealed dynamic features of structural regions in GPCRs that are not observable in the crystal structures. Thus, CXMS analysis of GPCRs using the expanded toolkit would facilitate structural modeling of uncharacterized receptors and gain new insights into receptor-ligand interactions.