Center for Integrated Protein Science (CIPSM) at the, Department of Chemistry, Technische Universität München, Lichtenbergstr. 4, 85747, Garching, Germany.
Physics Department and Institute for Advanced Study, Technische Universität München, Am Coulombwall 4a, 85748, Garching, Germany.
Angew Chem Int Ed Engl. 2019 May 20;58(21):7127-7132. doi: 10.1002/anie.201901056. Epub 2019 Apr 17.
The proteolytic complex ClpXP is fundamental to bacterial homeostasis and pathogenesis. Because of its conformational flexibility, the development of potent ClpXP inhibitors is challenging, and novel tools to decipher its intricate regulation are urgently needed. Herein, we present amino acid based phenyl esters as molecular probes to study the activity and oligomerization of the ClpXP complex of S. aureus. Systematic screening of (R)- and (S)-amino acids led to compounds showing potent inhibition, as well as stimulation of ClpXP-mediated proteolysis. Substoichiometric binding of probes arrested ClpXP in an unprecedented heptamer-hexamer assembly, in which the two heptameric ClpP rings are dissociated from each other. At the same time, the affinity between ClpX and ClpP increased, leading to inhibition of both enzymes. This conformational arrest is beneficial for the consolidated shutdown of ClpXP, as well as for the study of the oligomeric state during its catalytic cycle.
蛋白酶体复合物 ClpXP 是细菌内稳态和发病机制的基础。由于其构象灵活性，开发有效的 ClpXP 抑制剂具有挑战性，因此迫切需要新的工具来破译其复杂的调控机制。在此，我们提出了基于氨基酸的苯酯作为研究金黄色葡萄球菌 ClpXP 复合物活性和寡聚化的分子探针。（R）-和（S）-氨基酸的系统筛选导致了具有强抑制作用以及刺激 ClpXP 介导的蛋白水解作用的化合物。探针的亚化学计量结合将 ClpXP 固定在一种前所未有的七聚体-六聚体组装中，其中两个七聚体 ClpP 环彼此解离。同时，ClpX 和 ClpP 之间的亲和力增加，导致两种酶都被抑制。这种构象捕获有利于 ClpXP 的完全关闭，也有利于研究其催化循环中的寡聚状态。
