Role of a substrate binding pocket in the amino terminal domain of <i>Mycobacterium tuberculosis</i> caseinolytic protease B (ClpB) in its function

<i>Mycobacterium tuberculosis</i> (<i>Mtb</i>), the causative agent of tuberculosis when infects the host encounters several stresses within the host, resulting in aggregation of its proteins. To resolve this problem <i>Mtb</i> uses chaperones to either repair the damage or degrade the aggregated proteins<i>. Mtb</i> caseinolytic protein B (ClpB) helps in the prevention of aggregation and also resolubilization of aggregated proteins in bacteria, which is important for the survival of <i>Mtb</i> in the host. To function optimally, ClpB associates with its co-partners DnaK, DnaJ, and GrpE. The role of N-terminal domain (NTD) of <i>Mtb</i> ClpB in its function is not well understood. In this context, we investigated the interaction of three substrate mimicking peptides with the NTD of <i>Mtb</i> ClpB <i>in silico</i>. A substrate binding pocket, within the NTD of ClpB comprising of residues L136, R137, E138, K142, R144, R148, V149, Y158, and Y162 forming an ɑ-helix was thus identified. The residues L136 and R137 of the ɑ-helix were found to be important for the interaction of DnaK to ClpB. Further, nine single alanine recombinant variants of the identified residues were generated. As compared to the wild-type <i>Mtb</i> ClpB all the <i>Mtb</i> ClpB variants generated in this study were found to have reduced ATPase and protein refolding activity indicating the importance of the substrate binding pocket in ClpB function. The study demonstrates that the NTD of <i>Mtb</i> ClpB is important for its substrate interaction activity, and the substrate binding pocket identified in this study plays a crucial role in this interaction. Communicated by Ramaswamy H. Sarma