Reconstitution and functional characterization of MmpL3, an essential transporter from Mycobacterium tuberculosis

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.