Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
EMBO J. 2012 Mar 21;31(6):1529-41. doi: 10.1038/emboj.2012.5. Epub 2012 Jan 27.
Mycobacterium tuberculosis (Mtb) contains two clpP genes, both of which are essential for viability. We expressed and purified Mtb ClpP1 and ClpP2 separately. Although each formed a tetradecameric structure and was processed, they lacked proteolytic activity. We could, however, reconstitute an active, mixed ClpP1P2 complex after identifying N-blocked dipeptides that stimulate dramatically (>1000-fold) ClpP1P2 activity against certain peptides and proteins. These activators function cooperatively to induce the dissociation of ClpP1 and ClpP2 tetradecamers into heptameric rings, which then re-associate to form the active ClpP1P2 2-ring mixed complex. No analogous small molecule-induced enzyme activation mechanism involving dissociation and re-association of multimeric rings has been described. ClpP1P2 possesses chymotrypsin and caspase-like activities, and ClpP1 and ClpP2 differ in cleavage preferences. The regulatory ATPase ClpC1 was purified and shown to increase hydrolysis of proteins by ClpP1P2, but not peptides. ClpC1 did not activate ClpP1 or ClpP2 homotetradecamers and stimulated ClpP1P2 only when both ATP and a dipeptide activator were present. ClpP1P2 activity, its unusual activation mechanism and ClpC1 ATPase represent attractive drug targets to combat tuberculosis.
结核分枝杆菌（Mtb）含有两个 clpP 基因，它们都是生存所必需的。我们分别表达和纯化了 Mtb ClpP1 和 ClpP2。虽然它们都形成了十四聚体结构并被加工，但它们缺乏蛋白水解活性。然而，在确定了能显著（>1000 倍）刺激某些肽和蛋白质的 ClpP1P2 活性的 N-封闭二肽后，我们能够重新构建一个有活性的、混合的 ClpP1P2 复合物。这些激活剂协同作用，诱导 ClpP1 和 ClpP2 十四聚体解离为七聚体环，然后重新组装形成活性 ClpP1P2 双环混合复合物。目前还没有描述类似的小分子诱导的涉及多聚体环解离和再组装的酶激活机制。ClpP1P2 具有胰凝乳蛋白酶和半胱氨酸蛋白酶样活性，ClpP1 和 ClpP2 在切割偏好上有所不同。调节型 ATPase ClpC1 被纯化并被证明可以增加 ClpP1P2 对蛋白质的水解，但不能增加肽的水解。ClpC1 不能激活 ClpP1 或 ClpP2 同源十四聚体，只有当存在 ATP 和二肽激活剂时，才会刺激 ClpP1P2。ClpP1P2 的活性、其不寻常的激活机制和 ClpC1 ATPase 代表了对抗结核病的有吸引力的药物靶点。
