Mycobacterium tuberculosis ClpC1: characterization and role of the N-terminal domain in its function.
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.
Kar
Narayani P
NP
Immunochemistry Laboratory, National Institute of Immunology, New Delhi, India.
Sikriwal
Deepa
D
Rath
Parthasarathi
P
Choudhary
Rakesh K
RK
Batra
Janendra K
JK
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004789:Enzyme Activation; D017353:Gene Deletion; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic
D001426:Bacterial Proteins; D002413:Cations, Divalent; C534073:ClpC1 protein, Mycobacterium tuberculosis; D004269:DNA, Bacterial; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D000251:Adenosine Triphosphatases