Abstract
The mycobacterial membrane protein large 3 (MmpL3) transporter is essential and 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. However, the mechanism that MmpL3 uses to facilitate the transport of fatty acids and lipidic elements to the mycobacterial cell wall remains elusive. Here, we report 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-electron microscopy (cryo-EM) and X-ray crystallography. Combined with calculated results from molecular dynamics (MD) and target MD simulations, we reveal 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.
Highlights
Tuberculosis (TB) is an airborne disease caused by the bacterium Mycobacterium tuberculosis
We found that membrane protein large 3 (MmpL3) is a promiscuous membrane protein, capable of binding a variety of lipids, including trehalose monomycolate (TMM), phosphatidylethanolamine (PE), phosphatidylglycerol, and cardiolipin, all with dissociation constants within the micromolar range [14]
MAGP, mycolyl arabinogalactan peptidoglycan; MD, molecular dynamics; multidrug-resistant TB (MDR-TB), multidrugresistant TB; MM-GBSA, molecular mechanics generalized Born surface area; MmpL, Results Cryo-EM structure of MmpL3 reconstituted in nanodiscs mycobacterial membrane protein large; MmpL3ND, MmpL3-nanodisc; MR, molecular replacement; OGNG, octyl glucose neopentyl glycol; Principal component analysis (PCA), principal component analysis; PE, phosphatidylethanolamine; particle mesh Ewald (PME), particle mesh
Summary
Tuberculosis (TB) is an airborne disease caused by the bacterium Mycobacterium tuberculosis. Mycobacterial membrane via the MmpL3 transporter, we here present 7 structures of M. smegmatis MmpL3, either alone or bound with lipid moieties, using single-particle cryo-electron. MAGP, mycolyl arabinogalactan peptidoglycan; MD, molecular dynamics; MDR-TB, multidrugresistant TB; MM-GBSA, molecular mechanics generalized Born surface area; MmpL, Results Cryo-EM structure of MmpL3 reconstituted in nanodiscs mycobacterial membrane protein large; MmpL3ND, MmpL3-nanodisc; MR, molecular replacement; OGNG, octyl glucose neopentyl glycol; PCA, principal component analysis; PE, phosphatidylethanolamine; PME, particle mesh. RMS, root mean square; RMSD, root mean square deviation; TB, tuberculosis; RMSF, root mean square fluctuation; TDM, trehalose dimycolate; TMD, target MD; TMM, trehalose monomycolate; T6D, trehalose 6-decanoate This MmpL3-nanodisc (MmpL3-ND) sample was further purified using a Superose 6 column to separate the MmpL3-ND complex from empty nanodiscs.
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