Structure-function analysis of MmpL7-mediated lipid transport in mycobacteria

Area of Science:

• Microbiology
• Molecular Biology
• Structural Biology

Background:

• Mycobacterial membrane protein Large 7 (MmpL7) is a Resistance-Nodulation-Division (RND) transporter essential for exporting the virulence lipid phthiocerol dimycocerosate (PDIM) in *Mycobacterium tuberculosis*.
• The function of MmpL7 in transporting other lipids, like phenolic glycolipid (PGL), and its unique structural and mechanistic properties within the MmpL family are not fully understood.

Purpose of the Study:

• To investigate the role of MmpL7 in transporting phenolic glycolipid (PGL) in *Mycobacterium bovis* BCG.
• To elucidate the structural features and potential energy-coupling mechanism of *M. tuberculosis* MmpL7 through *in silico* modeling.
• To identify key residues involved in PDIM transport and biosynthesis using functional complementation assays.

Main Methods:

• Utilized a null mutant of *Mycobacterium bovis* BCG lacking MmpL7 to study PGL transport.
• Generated an *in silico* structural model of *M. tuberculosis* MmpL7.
• Performed functional complementation of the *M. bovis* BCG *mmpL7* mutant with mutated *mmpL7* alleles.

Main Results:

• MmpL7 was found to be involved in the transport of PGL, a lipid also produced by hypervirulent *M. tuberculosis* strains.
• The *in silico* model revealed MmpL7 as a functional outlier lacking a canonical proton-relay sequence, suggesting a novel energy-coupling mechanism.
• The periplasmic porter domain 2 insert (PD2-insert) of MmpL7 is highly alpha-helical.
• Specific residues in transmembrane domains TM4 and TM10, and the PD2 domain insert, were identified as crucial for PDIM transport and/or biosynthesis.

Conclusions:

• MmpL7 plays a role in the transport of both PDIM and PGL, contributing to the virulence of *Mycobacterium* species.
• MmpL7 employs a unique mechanism for transport, distinct from other RND transporters.
• Identification of critical residues provides insights into the structure-function relationship of MmpL7 and potential targets for therapeutic intervention.