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

Abstract

Mycobacterium tuberculosis (Mtb), 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 Mtb uses chaperones to either repair the damage or degrade the aggregated proteins. Mtb 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 Mtb in the host. To function optimally, ClpB associates with its co-partners DnaK, DnaJ, and GrpE. The role of N-terminal domain (NTD) of Mtb ClpB in its function is not well understood. In this context, we investigated the interaction of three substrate mimicking peptides with the NTD of Mtb ClpB in silico. 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 Mtb ClpB all the Mtb 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 Mtb 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

Keywords:

• Heat shock proteins
• TB
• stress
• chaperone
• protein folding
• mycobacteria

Author contributions

DS, PT, and JKB conceived the study. DS, RS, and PT performed the experiments. PT and JKB analyzed the data and wrote the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was partly supported by grant no. EMR/2017/001380 from the Scientific and Engineering Research Board (SERB), Govt. of India to JKB. JKB thankfully acknowledges a J. C. Bose Fellowship from SERB. DS acknowledges the Council of Scientific and Industrial Research, Govt. of India for a Junior Research Fellowship.