Schematic of a mycobacterial outer membrane

composed of mycolic acids (MAs), trehalose di- and mono-mycolate (TDM and TMM), phthiocerol dimycocerosate (PDIM), sulphoglycolipids (SGL), and pentacyl and diacyl trehalose (PAT and DAT). This membrane is covalently attached to the peptidoglycan via the arabinogalactan.

Chemical structures of mycobacterial lipids and membranes snapshots.

A. Chemical structure of α-mycolic acid (α-MA) with 78 carbons. B-D. Initial three-dimensional (3D) conformations (left) and snapshots after 3 μs production of pure mycolic acid bilayers (right) with fully extended (MA_eU), semi-folded (MA_eZ), and fully folded (MA_W) conformations. Lipids in the upper leaflet are colored in light, medium, and dark blue, and lipids in the lower leaflet are colored in white. Oxygen atoms are shown as van der Waals spheres. E-J. Chemical structures (left) and initial conformations (right) of the six outer leaflet lipids. K. Snapshot after 3 μs production of the symmetric system containing outer leaflet lipids at 313 K (All_Lipids_313). L. Snapshot after 3 μs production of the whole mycobacterial outer membrane with asymmetry of lipids at 313 K (Asym_313 system). Lipid colors in K and L match the colors of the initial 3D conformations.

Folding patterns and organization of mycomembrane inner leaflet symmetric bilayers.

A, D, G. Overlaid structures of one mycolic acid (A. MA_eU, B. MA_eZ, and C. MA_W) from every 10 frames of the final 500 ns production. Structures were aligned on 15 carbons at the top of the α-alkyl chain and 3 carbons at the top of the β-hydroxy chain: named C10 to C27 in the MA topologies. Oxygen atoms are displayed as red spheres. B, E, H. Lipid dynamics at 313 K (solid lines) and 333 K (dashed lines). The three columns are terminal carbon density profiles, average lipid order parameters of carbons32, and cyclopropane density profiles in 2 Å-wide slabs along the z-axis (i.e., the membrane normal) with the bilayer center at z=0. For carbon density profiles, lipids belonging to the lower leaflet are depicted in grey and those from the upper leaflet are depicted in blue. C, F, I. Snapshots with varying initial compositions after 3 μs production. Lower leaflet lipids are shown in white, upper leaflet lipids are shown in light, medium, and dark blue. Oxygen atoms are displayed as red spheres. Terminal carbons and cyclopropane carbons are shown as spheres colored according to their leaflet.

Phase transition of pure fully-extended α-mycolic acid bilayers.

A. Lateral diffusion coefficients from 313 K to 353 K. B. Terminal carbon (C75) z-density profiles separated by leaflet along the temperature gradient. Upper leaflet lipids are shown with solid lines, and lower leaflet lipids shown with dashed lines. C. Average order parameters of MA at varying temperatures. D. Cyclopropane carbons (CC1, CC2) z-density profiles along the temperature gradient. E. Snapshots along the temperature gradient, with lower leaflet lipids shown in white, upper leaflet lipids shown in colored lines, and oxygen atoms shown as spheres. Line and shaded area colors in B, C, and D correspond to those of upper leaflet lipids in E.

Lipid aggregation in mycomembrane outer leaflet symmetric bilayers.

A, C. Snapshots of All_Lipids systems after 3 μs production at 313 K and 333 K, respectively (see Supplementary Table 1 for system name, composition, and basic statistics). Lipid colors match the coloring from Fig. 2. Water and ions are omitted for clarity. B, D. Headgroup z position time series of PDIM (purple) and PAT (green) at 313 K and 333 K, respectively. Each line is a separate lipid molecule. E. Lateral diffusion coefficients from All_Lipids system for each lipid type. Solid bars are from the 313 K system, and hatched bars are from the 333 K system. Error bars are the standard errors across 3 replicas. F. Comparison of average deuterium order parameters of carbons along the z-axis for PDIM (light purple) and PAT (light green) at 313 K (solid lines) and 333 K (dashed lines). Shaded regions are the standard errors across 3 replicas. All analysis is averaged over the last 500 ns production.

Vertical heterogeneity and lipid dynamics in asymmetric mycomembranes.

A, C. Snapshots after 3 μs production at 313 K and 333 K, respectively. Lipid colors match the coloring from Fig. 2, and lipids are shown with the QuickSurf drawing method. B, D. Comparison of average deuterium order parameters of carbons along the z-axis for MA_eU (light blue), PDIM (light purple), and PAT (light green) at 313 K and 333 K, respectively. Solid lines are from the symmetric systems and dashed lines are from the asymmetric systems. E, G. Full lipid z-density profiles for each lipid at 313 K and 333 K, respectively. Shaded regions are the standard errors across 3 replicas. F, H. Headgroup z position time series of MA_eU (light blue), PDIM (light purple), and PAT (light green) at 313 K and 333 K, respectively. Each line is a separate lipid molecule. All analysis is averaged over the last 500 ns of production.