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Structure of an endogenous mycobacterial MCE lipid transporter

Nature volume 620, pages 445–452 (2023)Cite this article

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Abstract

To replicate inside macrophages and cause tuberculosis, Mycobacterium tuberculosis must scavenge a variety of nutrients from the host1,2. The mammalian cell entry (MCE) proteins are important virulence factors in M. tuberculosis1,3, where they are encoded by large gene clusters and have been implicated in the transport of fatty acids4,5,6,7 and cholesterol1,4,8 across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and it remains unclear how the approximately ten proteins encoded by a mycobacterial mce gene cluster assemble to transport cargo across the cell envelope. Here we report the cryo-electron microscopy (cryo-EM) structure of the endogenous Mce1 lipid-import machine of Mycobacterium smegmatis—a non-pathogenic relative of M. tuberculosis. The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex that is long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Our structural data revealed the presence of a subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated lipid import across the mycobacterial cell envelope.

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Fig. 1: Construction and purification of MceG–GFP.
Fig. 2: Cryo-EM structure of the endogenous MCE complex.
Fig. 3: The architecture of the portal and needle domains of the Mce1 system.
Fig. 4: Architecture of the ring and ABC transporter complex at the IM.
Fig. 5: LucB is an accessory factor that binds to the Mce1 complex.
Fig. 6: Model for Mce1-mediated transport.

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Data availability

The cryo-EM maps have been deposited at the Electron Microscopy Data Bank under the following accession codes: Map0 (EMD-29025), Map0a (EMD-29228), Map0b (EMD-29229), Map0c (EMD-29230), Map0d (EMD-29231), Map0e (EMD-29232), Map1 (EMD-29023), Map1a (EMD-29233), Map1b (EMD-29234), Map1c (EMD-29235), Map1d (EMD-29236), Map1e (EMD-29237), Map2 (EMD-29024), Map2a (EMD-29238), Map2b (EMD-29239), Map2c (EMD-29240), Map2d (EMD-29241) and Map2e (EMD-29242). The coordinates of the atomic models have been deposited at the PDB under the following accession codes: 8FEF (Map0), 8FED (Map1) and 8FEE (Map2). Coordinates for the atomic models used to make figures were obtained from the PDB under the following accession codes: 6XBD (MlaFEDB), 6MIT (LptBFGC), 2R19 (LptA), 5IV9 (LptDE), 5NIK (MacAB-TolC), 6V0D (LetB) and 1THQ (PagP). Cryo-EM data were deposited in Electron Microscopy Public Image Archive (11343). The MS files are available at MassIVE under dataset identifier MSV000090807 and ProteomeXchange under identifier PXD038456. A list of the bacterial strains and plasmids that have been deposited at Addgene is provided in Supplementary Table 1 with their identifiers.

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Acknowledgements

We thank the members of the Bhabha and Ekiert laboratories for discussions; N. Coudray, J. Ilmain, G. Isom, M. Macrae, F. Rubino and J. Sudar for feedback on our manuscript; H. Darwin for supplying the M. smegmatis strain (mc2155); J. Cox, C. Vieni, K. Murphy, R. Voorhees and D. Görlich for sharing plasmids; the members of the Foley laboratory (Memorial Sloan Kettering Cancer Center) for providing purified rabbit GFP antibody; the members of the Chng laboratory (National University of Singapore) for discussion on the palmitate growth assay; K. Dancel-Manning for the illustration of the mycobacterial cell envelope; F. Lang and K. Dancel-Manning for overseeing the use of the Talos L120C microscope and the facility in which the Talos L120C is housed; A. Paquette, W. Rice and B. Wang for assistance with cryo-EM grid screening and microscope operation; the members of the Central Lab Services team at NYU School of Medicine for preparation of medium and buffers; and S. Mulligan and L. Vega for assistance with cryo-EM data collection. EM data processing has used computing resources at the HPC Facility at NYU, and we thank the members of the HPC team for high-performance computing support. This work was supported by the following funding sources: Schmidt Science Fellows (to J.C.), Jane Coffin Childs Memorial Fund for Medical Research (to J.C.), pilot funding from the NYU Langone Health Antimicrobial-resistant Pathogen Program (to G.B. and D.C.E.) and NIH/NIAID 1R01AI174646 (to G.B. and D.C.E.). G.B. is a Pew Scholar in the Biomedical Sciences, supported by The Pew Charitable Trusts (PEW-00033055). The NYU Microscopy Center is partially supported by NYU Cancer Center Support Grant NIH/NCI P30CA016087. The MS experiments were supported in part by NYU Grossman School of Medicine and with a shared instrumentation grant from the NIH (1S10OD010582-01A1) for the purchase of an Orbitrap Fusion Lumos. A portion of this research was supported by NIH grant U24GM129547 and performed at the PNCC at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research.

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Authors and Affiliations

  1. Department of Cell Biology, New York University School of Medicine, New York, NY, USA

    James Chen, Alice Fruhauf, Catherine Fan, Gira Bhabha & Damian C. Ekiert

  2. Proteomics Laboratory, Division of Advanced Research Technologies, New York University School of Medicine, New York, NY, USA

    Jackeline Ponce & Beatrix Ueberheide

  3. Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, USA

    Beatrix Ueberheide

  4. Department of Neurology, New York University School of Medicine, New York, NY, USA

    Beatrix Ueberheide

  5. Department of Microbiology, New York University School of Medicine, New York, NY, USA

    Damian C. Ekiert

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Contributions

J.C., D.C.E. and G.B. conceived the project. D.C.E. and G.B. supervised and administered the project. J.C. performed cloning, protein purifications and biochemistry. J.C. prepared cryo-EM samples, collected and processed cryo-EM data. J.C., D.C.E. and G.B. built models and performed structural analysis. J.C., A.F. and C.F. performed phenotypic assays. J.P. and B.U. performed MS experiments and analyses. J.C., B.U., D.C.E. and G.B. acquired funding for the project. J.C., D.C.E. and G.B. wrote the original draft of the manuscript. J.C., A.F., C.F., B.U., D.C.E. and G.B. revised and edited manuscript.

Corresponding authors

Correspondence to Gira Bhabha or Damian C. Ekiert.

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Extended data figures and tables

Extended Data Fig. 1 MCE systems in Mycobacterium tuberculosis and Mycobacterium smegmatis.

a, Schematic of the four Mammalian Cell Entry (MCE) operons in the Mycobacterium tuberculosis (Mtb) genome. Arrows represent individual genes and their direction. Genes coloured as indicated the key below. Operons annotated with an asterisk are conserved between Mtb and Mycobacterium smegmatis (Msmeg). b, Schematic of the six MCE operons in the Msmeg genome. Genes coloured as in Extended Data Fig. 1a. c, Size exclusion chromatogram of MceG-GFP purification (green) superimposed with protein standards (grey dotted line). Black arrow indicates protein sample shown in Fig. 1f and analysed by mass spectrometry in Fig. 1g.

Extended Data Fig. 2 Cryo-EM data processing workflow (Part 1).

a, Cryo-EM data processing pipeline. b, Representative cryo-EM micrograph from 43,925 micrographs. Particles of interest are circled in white. Scalebar (100 nm) is indicated on the bottom right of the micrograph. Three biologically independent grid preparations of purified MceG-GFP (n = 3) yielded similar micrographs. c, Representative 2D classes of complex. Eight 2D class averages showing different views of the particles were generated in cryoSPARC52 using the final set of ‘consensus’ particles extracted with box size of 360 pixels and no binning.

Extended Data Fig. 3 Cryo-EM data processing workflow (Part 2).

a, Processing pipeline for local refinements performed in cryoSPARC52 and composite map generation. Locally refined maps and raw map for consensus set of particles are shown. Locally refined maps for the consensus set of particles were contoured with the following levels: Map0a (0.281), Map0b (0.257), Map0c (0.259), Map0d (0.199), Map0e (0.17) in ChimeraX53. Map labels are indicated above the map and particle count and average resolution as reported by cryoSPARC are shown below. Composite density maps were generated in PHENIX64. b, Composite Map0 coloured by local resolution that was estimated in cryoSPARC. (left) Whole structure view. Map0 contoured to 12.7. (right) Cross-sectional view. (bottom) key for local resolution colouring, ranging from 2.30 Å (blue) to 4.10 Å (red). c, Gold-standard FSC curve calculated in cryoSPARC for composite, locally refined, and raw maps for consensus set of particles. The dotted line represents the 0.143 FSC cutoff. d, Directional 3DFSC63 calculated for Map0 (composite). e, Locally refined and raw maps for Class 1. Locally refined maps for Class 1 were contoured with the following levels: Map1a (0.172), Map1b (0.201), Map1c (0.185), Map1d (0.167), Map1e (0.15). Map labels are indicated above the map and particle count and average resolution as reported by cryoSPARC are shown below. f, Composite density map for Class 1 (Map1) coloured by local resolution that was estimated in cryoSPARC. (left) Whole structure view. Map1 contoured to 10.1. (right) Cross-sectional view. (bottom) key for local resolution colouring, ranging from 2.30 Å (blue) to 4.10 Å (red). g, Gold-standard FSC curve calculated in cryoSPARC for composite, locally refined, and raw maps for Class 1. The dotted line represents the 0.143 FSC cutoff. h, Directional 3DFSC calculated for Map1 (composite). i, Locally refined and raw maps for Class 2. Locally refined maps for Class 2 were contoured with the following levels: Map2a (0.177), Map2b (0.148), Map2c (0.163), Map2d (0.126), Map2e (0.15). Map label is indicated above the map and particle count and resolution are shown below. j, Composite density map for Class 2 (Map2) coloured by local resolution that was estimated in cryoSPARC. (left) Whole structure view. Map2 contoured to 10.2. (right) Cross-sectional view. (bottom) key for local resolution colouring, ranging from 2.30 Å (blue) to 4.10 Å (red). k, Gold-standard FSC curve calculated in cryoSPARC for composite, locally refined, and raw maps for Class 2. The dotted line represents the 0.143 FSC cutoff. l, Directional 3DFSC calculated for Map2 (composite).

Extended Data Fig. 4 Model to map fits and representative density from areas at different resolutions.

a, Examples of model to map fits comparing Mce1 (green) and Mce4 (red) proteins fit into our density. Proteins are shown as sticks and superimposed on to the cryo-EM density from Map0, shown as a transparent grey surface. Protein densities rendered using ChimeraX53 ‘volume zone’ with 2.0-3.0 Å distance cutoff around the indicated protein residues with the following contour levels: YrbE1A (12.0); YrbE1B (10.0) Mce1A/oMce1A (10.0), Mce1B (10.0), Mce1C (10.0), Mce1D (10.0), Mce1E (10.0), Mce1F (8.0). Red asterisks highlight examples of key residues that differ between Mce1 and Mce4 proteins, which led to the identification of Mce1 as the predominant complex represented in our cryo-EM density. b, Examples from each subunit for Map0. Examples are arranged left to right from lower to higher resolution. Protein densities rendered using ChimeraX53 ‘volume zone’ with 2.0 Å distance cutoff around the indicated protein residues with the following contour levels: Mce1A/oMce1A (6.0), Mce1F (14.0), Mce1E (10.0), MceG protomer 2 (10.0), Mce1C (8.0), MceG protomer 1. YrbE1A (12.0), Mce1D (8.0), Mce1B (8.0), YrbE1B (10.0). c, Examples of ligand density from Map0. Ligand densities rendered using ChimeraX ‘volume zone’ with 2.5 Å distance cutoff around UNLs and with the following contour levels: UNL1 (8.0), UNL4 (6.0), UNL20 (8.0). d, Examples of fits for LucB transmembrane helices (labeled TM1-TM4) from Map1. Protein densities rendered using ChimeraX ‘volume zone’ with 2.5 Å distance cutoff around each helix of LucB and contour level 7.0.

Extended Data Fig. 5 Final Mce1 model and comparison with periplasm-spanning transporters in double-membraned bacteria.

a, Missing regions in consensus composite density map (Map0). Map0 contoured to 10.0 and Map0 colour-coded based on the key on the right. Regions of the proteins that were not resolvable in the cryo-EM map are indicated by dotted lines, drawn approximately to scale, and indicated in the chart. b, Protein complexes that span the cell envelope in Gram-negative bacteria. (left) LPS exporter (modelled based on PDBs 6MIT23, 2R1977, 5IV978); (middle) MacAB-TolC efflux pump (PDB 5NIK79); (right) LetAB transporter (PDB 6V0D80). c, Structure of mycobacterial Mce1 transporter.

Extended Data Fig. 6 Structural features of Mce1ABCDEF portal and needle modules in the Mce1 complex.

a, Schematic of Mce1ABCDEF proteins showing boundaries for the transmembrane helix (TM), MCE domain (MCE), eight needle modules, and portal domain/C-terminus. b, C-termini of Mce1ABCDEF that form the portal domain, viewed as indicated by inset. Proteins are shown as transparent molecular surfaces and coloured as in the key. Superimposed in each panel are the residues of the indicated protein, shown as cartoon. c, Alignment of E. coli PagP (PDB 1THQ)37 and the Mce1 six-stranded β-barrel of the portal. Proteins were aligned in ChimeraX53. PagP is coloured grey and the Mce1 six-stranded β-barrel is coloured purple. d, Structural alignment of the eight structural modules in PyMOL using ‘cealign’ command (Schrödinger, LLC.). Proteins are superimposed, and coloured as in Extended Data Fig. 6a. e, Hydrophobicity and radius of tunnel in Mce1 needle calculated using MOLE 2.574. X-axis shows distance going down the tunnel (from left to right); Y-axis shows the distance away from the central axis of the tunnel. Plot shows hydrophobicity, where blue is hydrophilic and yellow is hydrophobic.

Extended Data Fig. 7 Structural features of MCE domains and ABC transporter transmembrane domains, YrbE1A and YrbE1B.

a, Structural alignment of Mce1 MCE domains colour-coded based on key. Domains were aligned to the MCE domain of Mce1A. Pore-lining loops (PLLs) are circled. b, Protein sequence alignment of the MCE domains from Msmeg Mce1 proteins using MUSCLE75 and visualized using JalView76. Sequence alignment is coloured by BLOSUM62 score (not conserved, white; conserved, blue). PLL region is highlighted with black bracket and pore facing residues are indicated with black arrows. c, Gallery of Mce1 PPLs. Protein backbones are shown as cartoon tubes with residues shown as sticks and coloured as Extended Data Fig. 7a. Cryo-EM density for the PLLs is shown as a grey transparent surface. Protein densities rendered using ChimeraX53 ‘volume zone’ with 2.0 Å distance cutoff around each PLL at contour level 10.0. Pore facing residues are annotated. d, Topology diagram of YrbE1A, Yrbe1B and E. coli homologue MlaE (derived from PDB 6XBD41). CH, coupling helix; PH, periplasmic helix; IF, interfacial helix; TM, transmembrane helix. e, Structures of YrbE1A, YrbE1B and MlaE. YrbE1A and YrbE1B form a heterodimer, while MlaE forms a homodimer. One protomer is shown as a cartoon and the other as a molecular surface in each representation. f, View of Mce1 IM complex as indicated by inset on upper left. Model coloured as in the key. The C-terminus of YrbE1B (residues 280–289) is shown as spheres. Grey-dotted lines indicate the MCE ring tilt relative to YrbE1B. g, Zoom-in view of region boxed in Extended Data Fig. 7f, oriented as indicated by inset, highlighting interaction between YrbE1B C-terminus and Mce1F PLL. Proteins are shown as cartoon ribbons and coloured as Extended Data Fig. 7f. Cryo-EM density is shown as a transparent grey surface for the YrbE1B C-terminus (chain J, residues 280–289) and Mce1F PLL (chain F, residues 99–110). Protein densities rendered using ChimeraX53 ‘volume zone’ with 2.0 Å distance cutoff around YrbE1B C-terminus and Mce1F PLL and 8.7 contour level. Hydrogen bonds are shown as cyan dotted lines.

Extended Data Fig. 8 Protein expression of MceG, Mce1F, Mce4F for mceG bacterial strains used in growth assays.

a, Representative western blots showing protein levels of MceG, Mce1F, Mce4F in the lysates of mceG bacterial strains used in growth assays. Labels above blots denote the Msmeg genotype and mceG plasmid used for complementation and molecular weight markers (kDa) are indicated on the right. Lysates of Δmce1 and Δmce4 were loaded as controls for anti-Mce1F and anti-Mce4F antibody specificity. Lysates were derived from the same experiment and blots were processed in parallel. For gel source data, see Supplementary Fig. 1b. Western blots were performed with four independent biological replicates (n = 4), each with similar result. b, Plot showing fold change of protein levels of MceG (red), Mce1F (blue), Mce4F (yellow) in mceG mutants compared to Wild-type Msmeg (WT) based on the quantification of the western blots. Custom anti-MceG antibody was raised against the peptide 241-NGRRIGPIGMSEEKD-255, therefore cannot be used to detect MceG(Δ242-360) (denoted with black asterisk on plot). For gel source data, see Supplementary Figure 1b. Plotted data are the mean of four biological replicates (n = 4) and error bars representing standard deviations are shown.

Extended Data Fig. 9 LucB purification, proteomics, growth assay, and western blot results.

a, SDS-PAGE of LucB-GFP affinity purification (left) and Western blot using an anti-GFP antibody against purified LucB-GFP (right). For gel source data, see Supplementary Fig. 1c. Purifications were performed with three independent biological replicates (n = 3), with similar results. b, Size exclusion chromatogram of LucB-GFP purifications (pink line) superimposed with protein standards (grey dotted line). Black arrow indicates protein sample further analysed by negative stain electron microscopy (Extended Data Fig. 9c,d) and mass spectrometry (Extended Data Fig. 9e). c, Negative stain electron microscopy micrograph of purified LucB-GFP from 60 micrographs. Particles of interest, which resemble the Mce1 complex, are circled in white. Scalebar (200 nm) is indicated on the bottom left of the micrograph. Three biologically independent grid preparations of purified LucB-GFP (n = 3) yielded similar micrographs. d, 2D class average of ‘MCE-like’ particles from negative stain electron microscopy of endogenously purified LucB-GFP. Scale bar = 20 nm. e, Plot of proteins identified by mass spectrometry that co-purify with LucB-GFP. Each point corresponds to an individual protein plotted by fold change difference after purification of LucB-GFP from Msmeg strain harbouring tagged LucB versus control wild-type Msmeg mc2155 (x-axis) and the probability that a protein is a LucB interactor (SAINT score; y-axis). SAINT score = 1 identifies proteins with the highest probability of being a LucB interactor51. SAINT score ≥ 0.67 yielded an FDR (false discovery rate) of ≤ 5% as indicated by the purple dotted line. Proteins related to the mycobacterial MCE systems are highlighted and annotated as shown in the key. Plotted data are from three biological replicates. f, Growth curves in minimal media containing cholesterol as the sole carbon source for ΔlucB (pink), WT Msmeg mc2155 strain (black), ΔmceG (red), and Δmce4 (yellow). Plotted data are the mean of three biological replicates (n = 3) and error bars representing standard deviations are shown. g, Representative western blots showing protein levels of Mce1F and Mce4F in Msmeg strains as indicated in the figure. Labels above blots denote the Msmeg genotype and lucB plasmid used for complementation and molecular weight markers (kDa) are indicated on the right. Lysates were derived from the same experiment and blots were processed in parallel. For gel source data, see Supplementary Fig. 1d. Western blots were performed with three independent biological replicates (n = 3), each with similar result. h, Plot showing fold change of protein levels of Mce1F (blue) and Mce4F (yellow) in lucB bacterial strains compared to Wild-type Msmeg (WT) based on the quantification of the western blots. For gel source data, see Supplementary Figure 1d. Plotted data are the mean of three biological replicates (n = 3) and error bars representing standard deviations are shown.

Extended Data Fig. 10 LucB orthologs are found in other bacteria with MCE systems.

a, (left) 2D topology diagram of LucB. (right) Protein sequence alignment of LucB orthologs from fifteen different mycobacterial species generated using MUSCLE75 and visualized using JalView76. Sequence alignment is coloured by percent identity (not identical, white; identical, blue). Transmembrane helices of LucB (labeled TM1-TM4) are depicted above and residues that interact with Mce1C are highlighted below with asterisks. b, Circular phylogenetic tree showing spread of orthologs of LucB in other bacteria. LucB orthologs were compiled from eggNOG v5.081 and AlphaFold Protein Structure Database48. Protein sequences were aligned to generate a phylogenetic tree using MUSCLE75 and the tree was visualized in ITOL82. Leaves indicate individual species and are coloured by bacterial family: Mycobacteriaceae (red), Intrasporangiaceae (yellow), Pseudonocardiaceae (green), Tsukamurellaceae (cyan), Nocardiaceae (blue), Gordoniaceae (purple). Tree scale indicated on bottom right.

Supplementary information

Supplementary Information

Supplementary Tables 1, 4 and 6 and Supplementary Fig. 1 (uncropped gels and blots from Fig. 1 and Extended Data Figs. 8 and 9).

Reporting Summary

Peer Review File

Supplementary Table 2

Peptide spectral matches for M. smegmatis proteins that co-purified with MceG–GFP or LucB–GFP. MS analysis of purifications of MceG–GFP and LucB–GFP compared to WT mc2-155. Coverage, number of peptides and peptide spectral matches are indicated for each interacting M. smegmatis protein.

Supplementary Table 3

SAINT scores for M. smegmatis proteins that co-purified with MceG–GFP. M. smegmatis proteins that interact with MceG–GFP based on MS data of MceG–GFP purifications. SAINT scores indicating the probability that a given protein is a MceG interactor are shown.

Supplementary Table 5

SAINT scores for M. smegmatis proteins that co-purified with LucB–GFP. M. smegmatis proteins that interact with LucB–GFP based on mass spectrometry data of LucB–GFP purifications. SAINT scores indicating the probability that a given protein is a LucB interactor are shown.

Supplementary Video 1

Overview of Mce1 structure. Video highlighting the overall cryo-EM structure of Mce1 transporter and key structural features of the complex.

Supplementary Video 2

Model for Mce1-mediated transport. Video illustrating a model for how substrates are transported by Mce1 with potential steps annotated: (1) substrates enter the Mce1 portal domain; (2) substrates travel through the tunnel in the needle; (3) substrates pass through the MCE ring and enter the substrate binding pocket of the ABC transporter; and (4) ATP drives the transport of the molecules from the pocket into either the IM or cytoplasm.

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Chen, J., Fruhauf, A., Fan, C. et al. Structure of an endogenous mycobacterial MCE lipid transporter. Nature 620, 445–452 (2023). https://doi.org/10.1038/s41586-023-06366-0

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