Ecumicin is a well-known and potent inhibitor of Mycobacterium tuberculosis. Although the target of ecumicin is caseinolytic protease C1 (ClpC1), the exact mechanism by which ecumicin inhibits ClpC1 has not been identified. To analyze ecumicin's action on ClpC1, site-directed mutagenesis was performed on its binding site. The estimated binding residues within ClpC1 to ecumicin were selected via in silico analysis using molecular docking. The selected residues were mutated by site-directed mutagenesis and the effects on ecumicin binding were analyzed. Mutation at the R83 residue, especially the R83A mutation, in ClpC1 resulted in strong resistance to ATPase activation and inhibition of proteolytic activity. In addition, binding of ecumicin to the R83A ClpC1 N-terminal domain (residues 1-145) was not observed in native gel analysis. These results reveal that the R83 residue plays an important role in the binding of ecumicin. This result provides a basis for the development of an anti-tuberculosis agent based on ecumicin derivatives.
Keywords: Caseinolytic protease C1; Ecumicin; Molecular docking; Mycobacterium tuberculosis; Site-directed mutagenesis.
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