Role of DnaK in HspR‐HAIR interaction of Mycobacterium tuberculosis

Abstract

Heat shock proteins (Hsps) are a highly conserved family of proteins. The regulation of expression of Hsps in Mycobacterium tuberculosis, is regulated both positively and negatively by alternate sigma factors and transcriptional DNA repressors, respectively. HspR is a negative regulator of expression of hsps, DnaK, ClpB, and Acr2 in M. tuberculosis. In this study, we expressed the M. tuberculosis HspR (MtHspR) in E. coli, and functionally characterized it. MtHspR independently bound to its putative cognate DNA, the HAIR element. MtHspR was found to exist in a dynamic mixture of dimeric and monomeric protein and presence of salt led to the formation of trimers which lacked the DNA binding activity. MtHspR was found to be heat stable with a T m of 668C. HspR-HAIR binding was stable upto 608C suggesting that MtHspR is not the heat stress sensor. Mycobacterial DnaK was found to interact directly with MtHspR-HAIR complex in vitro in an ATP independent manner. The DnaK-HspR-HAIR binding pattern altered at high temperatures in the presence of aggregated a-casein substrate, suggesting that DnaK may indirectly be responding to heat stress in a feedback loop mechanism.

Loss-of-Function Mutations in HspR Rescue the Growth Defect of a Mycobacterium tuberculosis Proteasome Accessory Factor E ( pafE ) Mutant

“…HspR migrated as two major species. The first species eluted at the expected retention time for an HspR dimer, with a molecular mass of ϳ28 kDa, as has been reported elsewhere (23). The second species eluted later, with a retention time of 21.1 ml.…”

Chaperone addiction of toxin–antitoxin systems

“…The fact that TAC chaperones can replace the E. coli export chaperone SecB suggests that stress-induced protein aggregation or de novo synthesis of specific secretory proteins could hijack the chaperone and ultimately lead to the activation of the toxin for adaptive purposes12. When bound to the antitoxin, the chaperone could also act as a transcriptional co-repressor of TAC , similarly to what was previously described for the DnaK and GroEL chaperones, known to act as co-repressors of heat-shock genes by modulating the activity of the transcriptional repressors HspR and HrcA, respectively232425. Such a mechanism would ensure a robust repression of toxin synthesis and directly link toxin expression to specific changes that do not only rely on proteases activation26272829 but on chaperone availability as well12.…”

The amino‐terminal domain of Mycobacterium tuberculosis ClpB protein plays a crucial role in its substrate disaggregation activity

“…The co‐chaperone DnaK was purified in the same manner as described previously . For purifying DnaJ1, the plasmid encoding DnaJ1 was overexpressed in E. coli strain BL21.…”

“…The supernatant was then loaded onto a Ni-nitrilotriacetic acid column pre-equilibrated with buffer A. Subsequently, the The co-chaperone DnaK was purified in the same manner as described previously [59]. For purifying DnaJ1, the plasmid encoding DnaJ1 was overexpressed in E. coli strain BL21.…”