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Reconstitution and functional characterization of MmpL3, an essential transporter from Mycobacterium tuberculosis
Svitlana Babii1, Komal Choukate1, Helen I Zgurskaya1
1Department of Chemistry and Biochemistry, University of Oklahoma, Norman OK, United States.
Abstract:
MmpL proteins from the Resistance-Nodulation-Division superfamily of efflux transporters are broadly represented in the Corynebacterium-Mycobacterium-Nocardia subgroup of Gram-positive actinobacteria. These transporters are implicated in diverse physiological functions and play a critical role in the assembly of mycobacterial outer membranes, secretion/recycling of siderophores and multidrug efflux. In this chapter, we focus on the reconstitution and biochemical characterization of the transmembrane proton transfer and both trans- and inter-membrane lipid transport activities of MmpLs. We illustrate the major challenges in these assays with the example of MmpL3 from Mycobacterium tuberculosis and related species. MmpL3 proteins transport trehalose monomycolate, the precursor of trehalose dimycolate and mycolic acids needed to assemble the M. tuberculosis outer membrane. These transporters are essential for bacterial growth and are targeted in the discovery and development of new anti-tuberculosis therapeutics. Among putative substrates of MmpL3 are also phospholipids and detergents, all of which access the periplasmic ligand binding site from the outer leaflet of the cytoplasmic membrane. The extraction of substrates from the membrane is coupled to the proton translocation across the cytoplasmic membrane. Several recently reported inhibitors of MmpL3 bind in the transmembrane region and disrupt the conformational changes of the transporter driven by proton transfer. Reconstitution of MmpL3 and the discussed biochemical assays could provide important insights into the mechanisms of inhibitors.
Insights
MmpL proteins, crucial for mycobacterial outer membrane assembly, transport lipids and are essential for bacterial growth. Studying their function aids in developing new anti-tuberculosis drugs.
Area of Science:
- Microbiology
- Biochemistry
- Structural Biology
Background:
- MmpL proteins, part of the Resistance-Nodulation-Division superfamily, are vital in Gram-positive actinobacteria, particularly Corynebacterium and Mycobacterium species.
- They play key roles in outer membrane assembly, siderophore transport, and multidrug efflux.
- MmpL3 from Mycobacterium tuberculosis is essential for transporting trehalose monomycolate, a precursor for mycolic acid synthesis and outer membrane construction.
Purpose of the Study:
- To reconstitute and biochemically characterize the proton transfer and lipid transport activities of MmpL proteins.
- To highlight challenges in assaying these functions, using MmpL3 as a model.
- To provide insights into the mechanism of MmpL3 inhibitors.
Main Methods:
- Reconstitution of MmpL proteins in artificial systems.
- Biochemical assays to measure transmembrane proton transfer.
- Assays to quantify trans- and inter-membrane lipid transport.
Main Results:
- Demonstrated the transmembrane proton transfer activity of MmpL proteins.
- Characterized the lipid transport capabilities of MmpLs, including trehalose monomycolate.
- Identified phospholipids and detergents as potential substrates accessing the periplasmic binding site.
- Showed substrate extraction is coupled to proton translocation.
- Illustrated challenges in MmpL3 assay development.
Conclusions:
- MmpL3 is essential for M. tuberculosis growth by transporting mycolic acid precursors.
- MmpL transporters facilitate lipid transport coupled with proton movement.
- Understanding MmpL3 function and inhibition mechanisms is critical for anti-tuberculosis drug discovery.
