Department of Chemistry, Chair of Organic Chemistry II, Center for Integrated Protein Science (CIPSM), Technische Universität München, Lichtenbergstraße 4, 85748 Garching, Germany.
Department of Biociences, Center for Integrated Protein Science (CIPSM), Technische Universität München, Emil-Erlenmeyer-Forum 8, 85354 Freising, Germany.
Cell Chem Biol. 2019 Jan 17;26(1):48-59.e7. doi: 10.1016/j.chembiol.2018.10.007. Epub 2018 Nov 8.
Detection of dynamic protein-protein interactions within complexes and networks remains a challenging task. Here, we show by the example of the proteolytic ClpXP complex the utility of combined chemical cross-linking and mass spectrometry (XL-MS) to map interactions within ClpP and ClpX as well as across the enigmatic ClpX hexamer-ClpP heptamer interface. A few hot-spot lysines located in signature loops in ClpX were shown to be in proximity to several structural regions of ClpP providing an initial draft of the ClpX-ClpP interaction. Application of XL-MS further confirmed that Listeria monocytogenes ClpX interacts with the heterooligomeric ClpP1/2 complex solely via the ClpP2 apical site. Moreover, cellular interaction networks of human and bacterial proteases were elucidated via in situ chemical cross-linking followed by an antibody-based pull-down against ClpP. A subsequent mass spectrometric analysis demonstrated an up to 3-fold higher coverage compared with co-immunoprecipitation without cross-linker revealing unprecedented insight into intracellular ClpXP networks.
检测复合物和网络内的动态蛋白质-蛋白质相互作用仍然是一项具有挑战性的任务。在这里，我们以蛋白酶体 ClpXP 复合物为例，展示了结合化学交联和质谱（XL-MS）来绘制 ClpP 和 ClpX 内以及神秘的 ClpX 六聚体-ClpP 七聚体界面之间相互作用的实用性。已经证明，ClpX 中几个位于特征环中的热点赖氨酸与 ClpP 的几个结构区域接近，提供了 ClpX-ClpP 相互作用的初始草案。XL-MS 的应用进一步证实，李斯特菌 ClpX 仅通过 ClpP2 顶端位点与异源寡聚体 ClpP1/2 复合物相互作用。此外，通过原位化学交联，然后针对 ClpP 进行基于抗体的下拉，阐明了人类和细菌蛋白酶的细胞相互作用网络。随后的质谱分析显示，与没有交联剂的共免疫沉淀相比，覆盖率高达 3 倍，这揭示了对细胞内 ClpXP 网络的前所未有的了解。
