Structural elucidation of the hexameric MmpS4-MmpL4 complex from Mycobacterium tuberculosis

Area of Science:

• Structural biology
• Microbiology
• Biochemistry

Background:

• Mycobacterium tuberculosis possesses thirteen MmpL transporters involved in exporting various molecules.
• MmpL4 and MmpL5, with MmpS4 and MmpS5, are responsible for exporting the siderophore mycobactin and the drug bedaquiline.
• Previous studies determined the MmpL4 monomer structure, lacking a key coiled-coil domain.

Purpose of the Study:

• To determine the cryo-EM structure of the hexameric (MmpS4)3-(MmpL4)3 complex.
• To characterize the conformation of MmpL4 within the hexameric complex.
• To establish a workflow for cryo-EM studies of labile protein complexes.

Main Methods:

• Rational disulfide cross-linking guided by AlphaFold predictions.
• Single particle cryo-electron microscopy (cryo-EM).
• Structure determination of the hexameric MmpS4-MmpL4 complex.

Main Results:

• The cryo-EM structure of the (MmpS4)3-(MmpL4)3 complex revealed the coiled-coil domain of MmpL4.
• The coiled-coil domain protrudes into the periplasm at a ~60° angle.
• MmpL4 exhibits a distinct conformation in the hexamer compared to the monomer, with a periplasmic cavity and rearranged proton coupling residues.

Conclusions:

• The study provides the first structural insights into the MmpL4-MmpS4 complex.
• The findings elucidate the structural basis for mycobactin and bedaquiline export.
• The developed workflow enables structural studies of challenging, labile protein complexes.