Engineered MmpS4-MmpL4 disulfide cross-links based on AlphaFold2 prediction.

(A) AlphaFold2 prediction of the hexameric (MmpS4)3-(MmpL4)3 complex. The three protomers of MmpL4 are shown in surface presentation and colored petrol, grey and white. The MmpS4 protomers are shown as yellow cartoon. The boundary of the cytoplasmic membrane is indicated by grey lines. The locations of the transmembrane domains (TMD) and the periplasmic PN/PC domains are indicated. MmpL4 is predicted to form a large coiled-coil domain (CCD) extending into the periplasm. (B and C) Based on the AlphaFold2 model, eight double-cysteine mutants (#1-#8) were generated, which exhibit a close distance between MmpL4 and MmpS4. The original residues that were substituted by cysteines are shown as sticks. The mutants #2-#8 are expected to form spontaneous disulfide cross-links in the oxidative environment of the periplasm. (D) List of characterized double cysteine mutants. Whenever possible, the distances were measured between the γ-atoms of the respective residues of MmpS4 and MmpL4. In case of alanine and proline residues, the atom nearest to the γ-atom of the partner residue was used to measure the distance. (E) Non-reducing (left) or reducing (right) SDS-PAGE analysis of purified cysteine cross-linking mutants of MmpS4-MmpL4, stained by coomassie. The negative control cross-linking pair #1 was not expected to form a disulfide bond because it is located in the reducing environment of the cytosol. The negative control cross-linking pair #9 bears cysteines that are too far apart to form a disulfide bond.

Structure of the hexameric (MmpS4)3-(MmpL4)3 complex.

(A) Left panel; Cryo-EM map of the hexameric (MmpS4)3-(MmpL4)3 complex processed using C1 symmetry viewed along the membrane plane and contoured at 4σ. The detergent micelle and weak density for E. coli ACP are shown as white, transparent density. MmpL4 protomers are colored in petrol, grey and white, MmpS4 in yellow. Density for a tilted CCD formed out of two α-helical hairpins donated from MmpL4 protomers A (petrol) and B (white) is visible. (B) Schematic labeling of MmpL4 domains. (C) Structural model of the asymmetric (MmpS4)3-(MmpL4)3 complex shown as cartoon shown along the membrane plane (left). (D) Transmembrane helices of MmpL4 and MmpS4 viewed from the periplasm. The transmembrane helices of MmpL4 are numbered. (E) Superimposed MmpS4-MmpL4 monomers of asymmetrically processed (MmpS4)3-(MmpL4)3 asymmetric orientation of the α-helical hairpins. complex, showing the asymmetric orientaon of the α-helical hairpins.

Detergent binding in a deep cavity extending into the periplasmic domain of MmpL4.

(A) Structural model of hexameric (MmpS4)3-(MmpL4)3 complex processed using C1 symmetry. Three detergent molecules (β-DDM) bound to each of the MmpL4 protomers are depicted as red/orange spheres. (B) Enlarged view of the β-DDM binding site of chain A. Non-proteinaceaous density (contoured at 6.8σ) interpreted as β-DDM is depicted in orange. (C – E) Cavity analysis of monomeric MmpL4 (PDB ID: 9GI0) (C), trimeric MmpL4 (this work, PDB ID: 9SYJ) (D) and monomeric MmpL3 (PDB ID: 7NVH) (E) using the 3V tool. Cavities are shown in yellow and the membrane boundary is indicated. The bottom row shows the conserved DY-pairs and S309(MmpL4)/S288(MmpL3) viewed from the periplasm. Hydrogen bonds are indicated by dashed lines.

Systematic structural comparison of monomeric versus trimeric MmpL4.

(A and B) Previously determined monomeric MmpL4 colored in light blue (PDB ID: 9GI0) was superimposed onto trimeric MmpL4 colored in dark blue (chain A of structure obtained using C1 symmetry, PDB ID: 9SYJ). (B) Two slices along the membrane plane are shown from the periplasm onto the TMDs at the two heights indicated by the dashed rectangles in (A). (C) Cα-atom distance plot between monomeric and trimeric MmpL4. The respective TM helices and domains are indicated above. For three structural elements showing marked differences, the superimposed structures are shown as cartoon above the plot. No structural information was available for the CCD and the C-terminus. The overall RMSD is 2.14 Å over 722 residues.

Channel prediction of MmpL4’s CCD.

(A) The CCD of trimeric MmpL4 as predicted by AlphaFold3 and shown as cartoon. A continuous channel leading through the CCD interior was calculated by the MOLEonline tool and shown as yellow surface. Methionine side chains lining the channel surface are shown as red sticks. Three top views along the pore are shown as insets. (B) Radius and biophysical properties of the channel shown in (A) calculated by the MOLEonline tool.

Cryo-electron microscopy data collection statistics

Nucleotide sequence of mmpS4-mmpL4_cys-depleted

Primers used in this study.

AlphaFold2 prediction of the hexameric (MmpS4)3-(MmpL4)3 complex.

(A) The three protomers of MmpL4 are shown in surface presentation and colored petrol, grey and white. The MmpS4 protomers are shown as yellow cartoon (only two of them are visible). The boundary of the cytoplasmic membrane is indicated by grey lines. (B) Predicted alignment error (PAE) plot of the hexameric (MmpS4)3-(MmpL4)3 complex reported by AlphaFold2. It shows the expected distance error (in Å) between each residue in the predicted model. The three MmpL4 protomers cover residues 1-2901, and the three MmpS4 protomers residues 2902-3321. (C) Predicted Local Distance Difference Test (pLDDT) plot. The three MmpL4 protomers cover residues 1-2901, and the three MmpS4 protomers residues 2902-3321. (D) Depiction of pLDDT values in the context of the predicted MmpL4/MmpS4 structure (only first protomer shown, N- and C-terminus of MmpL4 cleaved).

Size exclusion chromatograms.

(A-C) Size exclusion chromatography traces of wild-type MmpS4-MmpL4 (A), cys-depleted MmpS4-MmpL4 (B) and MmpS4-MmpL4 containing the cysteine pair #5 (C). The proteins were separated on a Superose 6 Increase 10/300 GL column. The main peak of MmpS4-MmpL4 containing the cysteine pair #5 eluting at 13.6 ml (indicated by red arrow) was concentrated and analyzed by cryo-EM. The cryo-EM structure of the hexameric (MmpS4)3-(MmpL4)3 complex was determined from this sample.

Functional analysis of cysteine depleted mutant.

(A) Viability of M. tuberculosis strains at increasing mycobactin concentrations was determined using the microplate Alamar Blue assay (see methods). Data were normalized to the viability in the absence of mycobactin (100 %). ΔL4L5, M. tuberculosis strain lacking mmpL4 and mmpL5; ::S4L4 complementation with mmpS4-mmpL4; ::S4L4_Cys complementation with cysteine depleted mmpS4-mmpL4. Data points correspond to technical triplicates. (B) Western blot analysis of MmpL4 extracted from the strains shown in (A) using an anti-3xFLAG antibody (see methods).

Cryo-EM reconstruction of the hexameric MmpS4-MmpL4 complex.

(A) Representative cryo-EM micrograph. (B) Image processing work flow. (C) 2D-class averages. (D) FSC plot used for resolution estimation. (E) Angular distribution plot of particles included in the final 3D reconstruction.

Local resolution estimations and cryo-EM densities of the hexameric MmpS4-MmpL4 complex.

(A) Final reconstructed maps colored by local resolution. Note that for the “long CCD” map, a large part of the map was not part of the mask and thus no local resolution could be determined (red parts, resolution arbitrarily set to zero). (B) Cryo-EM densities of the hexameric MmpS4-MmpL4 complex “short CCD” with the respective refined model superimposed. The model is shown as sticks and structural elements are labelled. Transmembrane helices (TM) are colored in green, MmpS4 in yellow. Densities were contoured at 5.4σ. (C) Cryo-EM densities of CCDs from “short CCD” and “long CCD” models, chains A on the left and chains B on the right. Densities were contoured at 4.2σ. Cryo-EM reconstruction of the hexameric MmpS4-MmpL4 complex.

Cryo-EM densities of DY-pairs.

The conserved DY-pairs and S309(MmpL4)/S288(MmpL3) are shown as sticks and viewed from the periplasm as in main Fig. 3. (A) Monomeric MmpL4 (PDB ID: 9GI0) contoured at 11.9σ. (B) Trimeric MmpL4 (this work, PDB ID: 9SYJ, chain A) contoured at 8.9σ. (C) Monomeric MmpL3 (PDB ID: 7NVH) contoured at 4.5σ.

Comparative analysis of CCDs.

(A) CCDs of M. tuberculosis MmpL proteins shown as cartoons. The models are AlphaFold3 predictions of the respective MmpS-MmpL hexamers (in case an MmpS protein is present) or the MmpL trimers. For each CCD, methionine residues are shown as red sticks and the number of methionine residues per α-helical hairpin is provided in the pictures on the left and in the pictures on the right, the models are colored according to their Predicted Local Distance Difference Test (pLDDT) values. The coloring scheme is provided at the bottom. (B) Phylogenetic tree of the CCDs shown in (A).

Structural comparisons of trimeric M. tuberculosis MmpL4 and MmpL5.

The first protomers of the trimeric MmpL4 structure (this study, PDB ID: 9SYJ, chain A) and the two trimeric MmpL5 structures (Xiong et al., PDB ID: 8ZKQ, chain A / Fountain et al., PDB ID: 9RFU, chain D) were superimposed using the matchmaker tool of ChimeraX (Needleman-Wunsch alignment algorithm, BLOSUM-62 similarity matrix). The respective RMDS values are depicted directly in the figure, along with the number of aligned residues.