MmpL3 is a lipid transporter that binds trehalose monomycolate and phosphatidylethanolamine

Abstract

The cell envelope of Mycobacterium tuberculosis is notable for the abundance of mycolic acids (MAs), essential to mycobacterial viability, and of other species-specific lipids. The mycobacterial cell envelope is extremely hydrophobic, which contributes to virulence and antibiotic resistance. However, exactly how fatty acids and lipidic elements are transported across the cell envelope for cell-wall biosynthesis is unclear. Mycobacterial membrane protein Large 3 (MmpL3) is essential and required for transport of trehalose monomycolates (TMMs), precursors of MA-containing trehalose dimycolates (TDM) and mycolyl arabinogalactan peptidoglycan, but the exact function of MmpL3 remains elusive. Here, we report a crystal structure of Mycobacterium smegmatis MmpL3 at a resolution of 2.59 Å, revealing a monomeric molecule that is structurally distinct from all known bacterial membrane proteins. A previously unknown MmpL3 ligand, phosphatidylethanolamine (PE), was discovered inside this transporter. We also show, via native mass spectrometry, that MmpL3 specifically binds both TMM and PE, but not TDM, in the micromolar range. These observations provide insight into the function of MmpL3 and suggest a possible role for this protein in shuttling a variety of lipids to strengthen the mycobacterial cell wall.

Keywords: MmpL3 transporter; X-ray crystallography; cell-wall biogenesis; mycobacterial membrane protein Large; native mass spectrometry.

Fig. 1.

Mass spectra of purified MmpL3 proteins. (A) Mass spectrum of full-length MmpL3 expressed in E. coli indicates that the protein exists as a monomer. Calculated and observed masses, including the C-terminal 6×His tag, are 110,222 and 110,246 Da. (B) Mass spectrum of full-length MmpL3 expressed in M. smegmatis. In addition to the monomeric protein in solution, the presence of degraded protein bands indicates that the purified protein is unstable. The two observed masses are 110,371 and 91,990 Da. (C) Mass spectrum of a 5-d-old sample of full-length MmpL3 expressed and purified from E. coli. The spectrum depicts three major degraded species in the solution sample. The observed masses of these species are 87,998, 84,842, and 83,359 Da, corresponding to residues 1–806, 1–776, and 1–763 of the protein, respectively. (D) Mass spectrum of the MmpL3₇₇₃ protein. The spectrum indicates that this protein exists as a monomer in solution. Observed and calculated masses in this case are 85,950 and 85,925 Da. The mass observed from the adduct peaks is 86,660 Da, which corresponds to the MmpL3₇₇₃-PE complex.

Fig. 2.

Structure of the M. smegmatis MmpL3 transporter. (A) Secondary structural topology of the MmpL3₇₇₃ monomer. The topology was constructed based on the crystal structure of MmpL3₇₇₃. The TMs are colored blue. The periplasmic loops 1 and 2 are in cyan and green, respectively. The CD of MmpL3 is colored red. (B) Ribbon diagram of a monomer of MmpL3₇₇₃ viewed in the membrane plane. The TMs, periplasmic loops 1 and 2, and CD are colored slate, cyan, green, and red, respectively. The bound DDM is depicted as yellow spheres, whereas the bound PE is depicted as pink spheres. The periplasmic loops 1 and 2 cross over each other to form the periplasmic domains 1 and 2 (PD1 and PD2). (C) The MmpL3₇₇₃ monomer forms a channel spanning the outer leaflet of the inner membrane up to the periplasmic domain. The orientation of this MmpL3 molecule has been rotated by 110° (as shown in the figure) compared with the orientation of B. The channel (colored gray) was calculated using the program CAVER (https://www.caver.cz/). The TMs, periplasmic loops 1 and 2, and CD are colored the same as in B. The bound DDM is depicted as yellow sticks, whereas the bound PE is depicted as pink sticks.

Fig. 3.

The DDM- and PE-binding sites of the MmpL3-PE complex. (A) The F_o − F_c electron density map of bound DDM in MmpL3. The bound DDM is shown as a stick model (yellow, carbon; red, oxygen). The F_o − F_c map is contoured at 3σ. Residues involved in DDM binding are shown as green sticks. The secondary structural elements of MmpL3₇₇₃ are colored light brown. (B) The F_o − F_c electron density map of bound PE in MmpL3. The bound PE is shown as a stick model (magenta, carbon; red, oxygen; blue, nitrogen). The F_o − F_c map is contoured at 3σ. Residues involved in PE binding are shown as green sticks. The secondary structural elements of MmpL3₇₇₃ are colored light brown.

Fig. 4.

Determination of dissociation constants for the binding of PE and TMM to MmpL3₇₇₃. (A) Mass spectra recorded for solutions of MmpL3₇₇₃ with increasing concentrations of PE. At 5 μM PE, a charge state series is observed (light brown), corresponding to bound PE, which increases in intensity as the PE concentration is increased to 80 μM. A second PE-binding peak (gray) emerges at concentrations above 20 μM. (B) Plot of relative fractional intensity of lipid-binding peaks over the total peak intensity versus PE concentration (Methods), yielding a curve for the first binding event and linear-like fit for the second, consistent with nonspecific PE binding. Each data point and SD are calculated from the average of five observed charge states in three independent experiments. Error bars represent SDs (n = 3). (C) Mass spectra recorded for solutions of MmpL3₇₇₃ with increasing concentrations of TMM. At 2.5 μM TMM, a charge state series is observed (orange), corresponding to bound TMM, which increases in intensity as the TMM concentration is increased to 40 μM. A second TMM-binding peak (purple) emerges at concentrations above 20 μM. (D) Plot of the relative fractional intensity of lipid-binding peaks over total peak intensity versus TMM concentration (Methods), yielding a curve for the first binding event and a linear-like fit for the second, consistent with nonspecific TMM binding. Each data point and SD are calculated from the average of five observed charge states in three independent experiments. Error bars represent SDs (n = 3).

Fig. 5.

Analysis of different components of purified M. smegmatis glycolipids and their binding to MmpL3₇₇₃. Mass spectrum of MmpL3₇₇₃ with purified M. smegmatis glycolipids shows a preferential binding to TMM (orange charge state series) but not to TDM. Purified lipid fraction has both TMM (red Inset) and TDM (blue Inset) at the lower m/z region.

Fig. 6.

Proposed mechanism for TMM translocation via MmpL3. This schematic diagram indicates that the MmpL3 transporter is capable of picking up a TMM molecule from the outer leaflet of the cytoplasmic membrane. This TMM molecule will pass through the channel formed by MmpL3 and arrive at the periplasmic lipid-binding site. The TMM moiety will then be exported to the inner leaflet of the outer membrane for the biosynthesis of TDMs and mAGPs.