2.A.6.5.6
The mycobacterial membrane protein large 3 (MmpL3) (Rv0206; 944 aas) may function with MmpL11 (TC# 2.A.6.5.5) (Tullius et al., 2011). MmpL3 exports trehalose monomycolate (TMM), involved in mycolic acid donation to the cell wall core (Tahlan et al., 2012). SQ109, a 1,2,-diamine related to ethambutol  is an inhibitor of MmpL3 (Tahlan et al., 2012).  The system may also transport heme.  Inhibitors have been identified (Rayasam 2013; Li et al. 2014).  MmpL3 has been shown to be a homotrimer of three 12 TMS subunits, confirming its RND-type structure (Belardinelli et al. 2016).  MmpL3 is a flipppase for mycolic acids, transporting them from the cytoplasmic side of the inner membrane to the external side. A 1.5-diarylpyrrole compound, BM212, is a potent inhibitor (Xu et al. 2017). Inactivation of the mmpL3 gene in M. neoaurum increased the permeability of the outer membrane and allowed increased uptake of sterols for coversion to other sterols for industrial purposes. One such product is 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC), used for the synthesis of various steroids in the industry (Xiong et al. 2017). Since iron deprivation decreases expression of the mmpL3 gene, a metal chelation strategy could boost the effectiveness of current anti-TB drug regimes to combat drug resistant TB (Pal et al. 2018). Crystal structures are available for MmpL3 alone and in complex with four TB drug candidates. MmpL3 consists of a periplasmic pore domain and a twelve-helix transmembrane domain. Two Asp-Tyr pairs centrally located in this domain appear to facilitate proton-translocation. SQ109, AU1235, ICA38, and rimonabant bind inside the transmembrane region and disrupt these Asp-Tyr pairs (Zhang et al. 2019). MmpL3 can be directly inhibited by several antitubercular compounds (Li et al. 2019). Yang et al. 2020 have determined crystal structures of MmpL3 in complex with NITD-349 and SPIRO. Both inhibitors bind deep in the central channel of the transmembrane (TM) domain and cause conformational changes to the protein. The amide nitrogen and indole nitrogen of NITD-349 and the piperidine nitrogen of SPIRO interact and clamp Asp645. Analysis of the two structures reveals that these inhibitors target the proton relay pathway to block the activity of MmpL3 (Yang et al. 2020). The  (MmpL3) transporter is required for shuttling the lipid trehalose monomycolate (TMM), a precursor of mycolic acid (MA)-containing trehalose dimycolate (TDM) and mycolyl arabinogalactan peptidoglycan (mAGP), in Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium smegmatis. It  facilitates the transport of fatty acids and lipidic elements to the mycobacterial cell wall. Su et al. 2021 reported 7 structures of the M. smegmatis MmpL3 transporter in its unbound state and in complex with trehalose 6-decanoate (T6D) or TMM using single-particle cryo-EM and X-ray crystallography. Combined with calculated results from molecular dynamics (MD) and target MD simulations, they revealed a lipid transport mechanism that involves a coupled movement of the periplasmic domain and transmembrane helices of the MmpL3 transporter that facilitates the shuttling of lipids to the mycobacterial cell wall (Su et al. 2021). Antibacterial compounds that target MmpL3 called ST004, have been identified and studied, showing that this compound strongly inhibits growth, and the cryoEM structure of MmpL3 with ST004 bound has been determined (Hu et al. 2022). PgfA (MSMEG_0317; TC# 1.B.163.1.9), a periplasmic protein that interacts with MmpL3, is a key determinant of polar growth and cell envelope composition in mycobacteria, and the LamA-mediated recruitment of this protein to one side of the cell is a required step in the establishment of cellular asymmetry (Gupta et al. 2022). Mycobacterial membrane protein large 3 (MmpL3) is an essential mycolic acid and lipid transporter required for growth and cell viability, and its function and inhibition have been reviewed (Williams and Abramovitch 2023). A continuous water pathway through the transmembrane region has been proposed, illustrating a putative pathway for protons. TMM can diffuse from the membrane into a binding pocket in MmpL3 spontaneously. Acetylation of TMM, which is required for transport, makes it more stable within MmpL3's periplasmic cavity compared to the unacetylated form (Li et al. 2023).  Equilibrium simulations revealed that trehalose monosmycolate can diffuse from the membrane into a binding pocket in MmpL3 spontaneously. Acetylation of TMM, which is required for transport, makes it more stable within MmpL3's periplasmic cavity (Li et al. 2023). Consistent with the close-open motion of the two PD domains, TMM entry size changes in the apo system, likely loading and moving the TMM, but it does not vary much in the holo system and probably impairs the movement of the TMM (Carbone et al. 2023). Water molecules passed through the central channel of the MmpL3 transporter to the cytoplasmic side in the apo system but not in the holo system, with a mean passing time of ∼135 ns. Because water wires play an essential role in transporting protons, these findings shed light on the importance of the PMF in driving the close-open motion of the two TMSs. The key channel residues involved in water passage display considerable overlap with conserved residues within the MmpL protein family, supporting their critical functional role (Carbone et al. 2023). The  inhibitory mechanism of anti-TB drug SQ109 involves the allosteric inhibition of TMM translocation by MmpL3 (Carbone et al. 2023).  Allosteric coupling of substrate binding and proton translocation in MmpL3 has been demonstrated (Babii et al. 2024).  It is required for the translocation of mycolic acids in the form of trehalose monomycolates (TMM) from the cytoplasm or plasma membrane to the periplasm or outer membrane (Babii et al. 2024).  MmpL3-dependent transport of TMM is essential for the growth of M. tuberculosis in vitro, inside macrophages, and in M. tuberculosis-infected mice. MmpL3 is also a validated target for several anti-mycobacterial agents. Coupled motions and varied conformational states likely contribute to the transport of TMM (Zhao et al. 2024), and simulations of inhibitor-bound MmpL3 showed an enlargement of the proton channel, potentially disrupting coupled motions. This indicates that inhibitors may impair MmpL3's transport function by directly blocking the proton channel, thereby hindering coordinated domain movements and indirectly affecting TMM translocation (Zhao et al. 2024).  MmpL11_TB and MmpL3_TB CTDs reveal notable features including a long unstructured linker that connects the globular domain to the last TMS in each transporter with charged lysine and arginine residues facing the membrane, and a C-terminal alpha helix (Berkowitz et al. 2024).  Novel indoles are antitubercular agents that simulate annealing-based analysis of their binding with MmpL3 (Ray et al. 2025).  Inhibitors of MmpL3 have also been identified (Eke et al. 2025). Enhancing inhibitor interactions with MmpL3, such as through hydrogen bonding or increasing inhibitor size to create larger physical barriers (e.g., interactions with Phe255 and Phe644), may prolong the inhibitors' residence times (Zhao et al. 2025).