Kar Narayani P, Sikriwal Deepa, Rath Parthasarathi, Choudhary Rakesh K, Batra Janendra K
Immunochemistry Laboratory, National Institute of Immunology, New Delhi, India.
FEBS J. 2008 Dec;275(24):6149-58. doi: 10.1111/j.1742-4658.2008.06738.x. Epub 2008 Nov 4.
Caseinolytic protein, ClpC is a general stress protein which belongs to the heat shock protein HSP100 family of molecular chaperones. Some of the Clp group proteins have been identified as having a role in the pathogenesis of many bacteria. The Mycobacterium tuberculosis genome demonstrates the presence of a ClpC homolog, ClpC1. M. tuberculosis ClpC1 is an 848-amino acid protein, has two repeat sequences at its N-terminus and contains all the determinants to be classified as a member of the HSP100 family. In this study, we overexpressed, purified and functionally characterized M. tuberculosis ClpC1. Recombinant M. tuberculosis ClpC1 showed an inherent ATPase activity, and prevented protein aggregation. Furthermore, to investigate the contribution made by the N-terminal repeats of ClpC1 to its functional activity, two deletion variants, ClpC1Delta1 and ClpC1Delta2, lacking N-terminal repeat I and N-terminal repeat I along with the linker between N-terminal repeats I and II, respectively were generated. Neither deletion affected the ATPase activity. However, ClpC1Delta1 was structurally altered, less stable and was unable to prevent protein aggregation. Compared with wild-type protein, ClpC1Delta2 was more active in preventing protein aggregation and displayed higher ATPase activity at high pH values and temperatures. The study demonstrates that M. tuberculosis ClpC1 manifests chaperone activity in the absence of any adaptor protein and only one of the two N-terminal repeats is sufficient for the chaperone activity. Also, an exposed repeat II makes the protein more stable and functionally more active.
酪蛋白水解蛋白ClpC是一种普遍应激蛋白，属于分子伴侣热休克蛋白HSP100家族。一些Clp家族蛋白已被确定在许多细菌的发病机制中起作用。结核分枝杆菌基因组显示存在ClpC的同源物ClpC1。结核分枝杆菌ClpC1是一种含848个氨基酸的蛋白质，在其N端有两个重复序列，并且包含所有被归类为HSP100家族成员的决定因素。在本研究中，我们对结核分枝杆菌ClpC1进行了过表达、纯化及功能特性分析。重组结核分枝杆菌ClpC1表现出内在的ATP酶活性，并能防止蛋白质聚集。此外，为了研究ClpC1的N端重复序列对其功能活性的贡献，我们分别构建了两个缺失变体ClpC1Delta1和ClpC1Delta2，它们分别缺失N端重复序列I以及N端重复序列I和N端重复序列II之间的连接区。这两种缺失均未影响ATP酶活性。然而，ClpC1Delta1的结构发生了改变，稳定性降低，并且无法防止蛋白质聚集。与野生型蛋白相比，ClpC1Delta2在防止蛋白质聚集方面更具活性，并且在高pH值和高温下表现出更高的ATP酶活性。该研究表明，结核分枝杆菌ClpC1在没有任何衔接蛋白的情况下表现出伴侣活性，并且两个N端重复序列中的仅一个对于伴侣活性就足够了。此外，暴露的重复序列II使该蛋白更稳定且功能上更具活性。
