Identifying key binding domain for the interaction of the Mycobacterium tuberculosis MmpL5 protein and bedaquiline/clofazimine
Objective To express and purify the MmpL5 protein of Mycobacterium tuberculosis so as to further investigate the interaction between MmpL5 protein and drugs and study the drug resistance mechanism caused by the MmpL5 efflux system.Methods The binding sites of the MmpL5 protein and drugs were predicted by molecular docking.Based on the results of molecular docking,the expression plasmid of the MmpL5-L1 truncated protein was constructed using the Mycobacterium tuberculosis H37Rv genome as a template.The MmpL5-L1 truncated protein was expressed and purified in Escherichia coli.The fluorescence quenching technique and the surface plasmon resonance technique were used to investigate the interaction between truncated proteins and drugs.Results MmpL5-L1(Q52-R202),a truncated protein of Mycobacterium tuberculosis,was successfully expressed and purified in Escherichia coli.The interaction between the truncated protein of MmpL5-L1 and clofazimine was validated by protein fluorescence quenching.The surface plasmon resonance experiment showed that the affinity constant(KD)of the truncated protein of MmpL5-L1 with bedaquiline was 4.033×10-5 and that with clofazimine was 1.218× 10-5.Conclusion The key amino acid region of MmpL5 binding to bedaquiline and clofazimine,which provided direct evidence for the binding of bedaquiline and clofazimine to MmpL5 protein and then efflux through the MmpS5-MmpL5 system,was successfully predicted and confirmed.This work laid a certain foundation for further elucidating the mechanisms of bedaquiline resistance and clofazimine cross-resistance.
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