Abstract
Mycobacterium tuberculosis ClpC1 is a member of the Hsp100/Clp AAA+ family of ATPases. The primary sequence of ClpC1 contains two N-terminal domains and two nucleotide binding domains (NBD). The second NBD has a long C-terminal sub-domain containing several motifs important for substrate interaction. Generally, ClpC proteins are highly conserved, however presence of C-terminal domains of variable lengths is a remarkable difference in ClpC from different species. In this study, we constructed deletion mutants at the C-terminus of M. tuberculosis ClpC1 to determine its role in the structure and function of the protein. In addition, a deletion mutant having the two conserved N-terminal domains deleted was also constructed to investigate the role of these domains in M. tuberculosis ClpC1 function. The N-terminal domains were found to be dispensable for the formation of oligomeric structure, and ATPase and chaperone activities. However, deletions beyond a specific region in the C-terminus of the ClpC1 resulted in oligomerization defects and loss of chaperonic activity of the protein without affecting its ATPase activity. The truncated mutants, defective in oligomerization were also found to have lost the chaperonic activity, showing the formation of oligomer to be required for the chaperonic activity of M. tuberculosis ClpC1. The current study has identified a region in the C-terminus of M. tuberculosis ClpC1 which is essential for its oligomerization and in turn its function.
Highlights
Molecular chaperones mediate correct folding, assembly, transport as well as disaggregation of proteins inside the cell during growth in normal as well as stressful conditions
We have investigated the role of C-terminus of ClpC1 of M. tuberculosis in its function
In Enterococcus faecalis, ClpB is linked to virulence [25], whereas ClpC has been associated with virulence in Bacillus subtilis and Listeria monocytogenes [10,11]
Summary
Molecular chaperones mediate correct folding, assembly, transport as well as disaggregation of proteins inside the cell during growth in normal as well as stressful conditions. The Hsp100 family consists of a group of AAA+ (ATPases associated with cellular activities) family of ATP-dependent chaperones that transfers misfolded proteins for degradation into the proteolytic chamber of an associated protease [5,6]. These proteases known as caseinolytic proteases or Clps are heterooligomers of an ATPase component belonging to the Hsp100 family and a protease component known as ClpP. Generally ClpC proteins are highly conserved subgroup within the Clp family, there are a few remarkable differences in different species One such difference is the presence of C-terminal domain of variable lengths. As compared to Clps of other bacteria, the M. tuberculosis ClpC1 has a long C-terminal extension beyond the conserved sensor 2/Box VIII (Figure 2). A detailed understanding of ClpC structure-function relationship has implications in exploring ClpC as a drug target in tuberculosis
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