Proton transfer activity of the reconstituted Mycobacterium tuberculosis MmpL3 is modulated by substrate mimics and inhibitors
Related Experiment Video
Updated: Sep 4, 2025
System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis
Published on: April 5, 2017
Casey M Stevens1, Svitlana O Babii1, Amitkumar N Pandya2
1Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019.
Abstract:
Transporters belonging to the Resistance-Nodulation-cell Division (RND) superfamily of proteins such as Mycobacterium tuberculosis MmpL3 and its analogs are the focus of intense investigations due to their importance in the physiology of Corynebacterium-Mycobacterium-Nocardia species and antimycobacterial drug discovery. These transporters deliver trehalose monomycolates, the precursors of major lipids of the outer membrane, to the periplasm by a proton motive force-dependent mechanism. In this study, we successfully purified, from native membranes, the full-length and the C-terminal truncated M. tuberculosis MmpL3 and Corynebacterium glutamicum CmpL1 proteins and reconstituted them into proteoliposomes. We also generated a series of substrate mimics and inhibitors specific to these transporters, analyzed their activities in the reconstituted proteoliposomes, and carried out molecular dynamics simulations of the model MmpL3 transporter at different pH. We found that all reconstituted proteins facilitate proton translocation across a phospholipid bilayer, but MmpL3 and CmpL1 differ dramatically in their responses to pH and interactions with substrate mimics and indole-2-carboxamide inhibitors. Our results further suggest that some inhibitors abolish the transport activity of MmpL3 and CmpL1 by inhibition of proton translocation.
Insights
Investigating Resistance-Nodulation-cell Division (RND) transporters like MmpL3 is crucial for antimycobacterial drug discovery. These transporters move key lipids, and their function can be inhibited by blocking proton translocation.
Area of Science:
- Biochemistry
- Molecular Biology
- Microbiology
Background:
- Resistance-Nodulation-cell Division (RND) superfamily transporters, including Mycobacterium tuberculosis MmpL3, are vital for Corynebacterium-Mycobacterium-Nocardia physiology.
- These transporters are critical drug targets for antimycobacterial therapies.
- They facilitate the transport of trehalose monomycolates, essential precursors for outer membrane lipid synthesis, via a proton motive force-dependent mechanism.
Purpose of the Study:
- To purify and functionally characterize MmpL3 and CmpL1 transporters.
- To investigate the interaction of substrate mimics and inhibitors with these transporters.
- To elucidate the mechanism of action of MmpL3 and CmpL1 inhibitors.
Main Methods:
- Purification of full-length and truncated MmpL3 and CmpL1 proteins from native membranes.
- Reconstitution of purified proteins into proteoliposomes.
- Analysis of transporter activity using substrate mimics and inhibitors.
- Molecular dynamics simulations of MmpL3 at varying pH levels.
Main Results:
- Reconstituted MmpL3 and CmpL1 proteins were shown to facilitate proton translocation across a phospholipid bilayer.
- Significant differences were observed in the pH response and substrate mimic interactions between MmpL3 and CmpL1.
- Indole-2-carboxamide inhibitors demonstrated specific interactions and affected transporter activity.
Conclusions:
- MmpL3 and CmpL1 exhibit distinct functional properties and responses to inhibitors.
- Some inhibitors function by disrupting the proton translocation activity of these essential transporters.
- These findings provide insights into the mechanism of RND transporters and inform the development of novel antimycobacterial drugs.
