Structure and assembly of the MmpL5/MmpS5 efflux transporter from Mycobacterium tuberculosis

Abstract

The MmpL5/MmpS5 efflux system in Mycobacterium tuberculosis plays crucial roles in extruding therapeutic drugs (e.g., bedaquiline), and exporting siderophores (i.e., (carboxy)mycobactins). However, the molecular basis underlying these processes remains unknown due to the lack of structural information. Here, we report the cryo-electron microscopy structures of Mycobacterium tuberculosis MmpL5/MmpS5 at resolutions ranging from 2.64 to 3.31 Å, revealing it to be a trimer. The core of this complex is formed by three MmpL5 subunits assembled in a unique shoulder-to-shoulder ring-like configuration, with each MmpS5 subunit positioned between the two adjacent MmpL5 subunits. A remarkable feature of this system is the extracellular stalk, which spans approximately 130 Å in length and is composed of three intertwined anti-parallel coiled-coil portions of MmpL5. The stalk secures the tight association of the three MmpL5 subunits and exhibits intrinsic structural flexibility. Additionally, an unexpected MmpL5 binder, AcpM, a mycobacterial acyl carrier protein, has also been identified. Collectively, the study provides insights into the biological assembly and molecular function of MmpL5/MmpS5, which will facilitate the discovery of innovative inhibitors for this system.

Fig. 1. Cryo-EM structure of the MmpL5-MmpS5-AcpM complex from Mycobacterium tuberculosis.

a Two views of the cryo-EM map of the MmpL5-MmpS5-AcpM complex colored by subunit (MmpL5, blue and silver; MmpS5, yellow; AcpM, pink). The volume threshold is 0.15 σ for the stalk and 0.3 σ for the other regions. b Cartoon representation of the MmpL5-MmpS5-AcpM complex structure from two different viewpoints. Color scheme same as in (a). A pseudo-atomic model of the stalk is also depicted and colored silver. Phosphatidylethanolamine (PE) and cardiolipin (CDL) molecules are shown as light green and orange sticks, respectively.

Fig. 2. Structure and characteristics of the MmpL5 trimer.

a Structural comparison of MmpL5 trimer with multidrug transporters AcrB (PDB code: 2DHH), MtrD (PDB code: 6VKS) and MexB (PDB code: 2V50). b Size exclusion chromatography (SEC) analyses of MmpL5 (L5), MmpL5/MmpS5 (L5/S5), and Stalk-truncated MmpL5/MmpS5 (L5_∆stalk/S5). Blue triangle, brown square and purple circle respectively indicate peaks 1, 2 and 3 in the SEC profiles. c BN-PAGE analyses of each peak as in (b). d Position of a pair of cysteine mutations introduced in the PC domains of MmpL5 (Left) and Western blot of the MmpL5 variant (MmpL5_A460C&A682C, Mut) and the purified wild-type MmpL5 trimer (Ctrl) (Right). Under non-reducing conditions, a high-molecular-weight band corresponding to the MmpL5 trimer was observed in the cysteine variant but not the wild-type MmpL5. In sharp contrast, this band migrated to a smaller molecular weight under reducing conditions. In (c, d), each experiment was repeated at least three times as independent biological replicates. Source data are provided as a Source Data file. e Superposition of MmpL5 (blue) and MmpL3 (gray; PDB code: 6AJF) seen from the plane of the membrane (Left) and a clipped view of the transmembrane region viewed from the periplasm (Right). f The Asp-Tyr pairs in TM4 and TM10 of MmpL5 and MmpL3 are shown as sticks. Hydrogen bonds are indicated as dashed lines. g, h Drug susceptibility data for MmpL5 variants. Error bars correspond to mean ± SD based on two independent biological replicates. The Gompertz model was used to fit growth data. Source data are provided as a Source Data file.

Fig. 3. Interplay between MmpL5 and MmpS5.

a The position of MmpS5 in the complex viewed from the cytoplasm. b Interactions between MmpS5 and MmpL5 in each protomer. Key residues are shown as sticks. Hydrogen bonds are indicated as dashed lines. c, d Close-up views of the membrane cleft between MmpS5 and two neighboring MmpL5 subunits. EM densities for PE and an unassigned ligand shown as surface with a volume threshold of 0.2 σ. The contact residues are shown as sticks. e, f Drug susceptibility data for three MmpL5/MmpS5 variants. Error bars correspond to mean ± SD based on two independent biological replicates. The Gompertz model was used to fit growth data. Source data are provided as a Source Data file.

Fig. 4. Interactions between MmpL5 and AcpM.

a Structure of MmpL5 bound AcpM represented in cartoon and surface. 4′-phosphopantetheine (Ppant) covalently linked to AcpM is shown as red sticks. b, c Close-up views of the binding site between MmpL5 and AcpM. Key residues are shown as stick models. Hydrogen bonds and salt bridges are represented by black dashed lines. d Superposition of M. smegmatis AcpM (pink) and M. tuberculosis MbtL (Alphafold 2 model, light green). Ppant and the modified residue (Ser41) of AcpM are shown as stick models with the electron density displayed as mesh at a 0.2 σ threshold. e Sequence alignment of AcpM and MbtL from M. smegmatis and M. tuberculosis. Contacting residues at the interface are marked by yellow spheres.