Braun's Lipoprotein Facilitates OmpA Interaction with the Escherichia coli Cell Wall

Abstract

Gram-negative bacteria such as Escherichia coli are protected by a complex cell envelope. The development of novel therapeutics against these bacteria necessitates a molecular level understanding of the structure-dynamics-function relationships of the various components of the cell envelope. We use atomistic MD simulations to reveal the details of covalent and noncovalent protein interactions that link the outer membrane to the aqueous periplasmic region. We show that the Braun's lipoprotein tilts and bends, and thereby lifts the cell wall closer to the outer membrane. Both monomers and dimers of the outer membrane porin OmpA can interact with peptidoglycan in the presence of Braun's lipoprotein, but in the absence of the latter, only dimers of OmpA show a propensity to form contacts with peptidoglycan. Our study provides a glimpse of how the molecular components of the bacterial cell envelope interact with each other to mediate cell wall attachment in E. coli.

Figure 1

Simulation setup. Given here is a snapshot of a simulation system with the full-length OmpA dimer (cyan and pink), BLP trimer (green), and PGN network (red). OmpA and BLP are embedded within an asymmetric bilayer containing Ra LPS in the upper leaflet (gray), and a mixture of phospholipids in the lower leaflet (yellow). To see this figure in color, go online.

Figure 2

BLP tilting and bending. (A) Shown here is the distribution of BLP tilt angle throughout the 100-ns simulations for systems without OmpA (black), with OmpA monomer (blue), and with OmpA dimer (red). This is measured as the acute angle between the centers of geometry of the phosphorus atoms in the inner leaflet of the OM, the N-terminal residues of the BLP, and its C-terminal residues (illustrated at the top of the graph). Independent repeats were plotted separately, and a bin size of 5° was used. (B) BLP helix kink angles were calculated along each of the three helices using VMD Bendix plugin (54). The figure shows the final snapshot of BLP from one of the BLP-only simulations, colored based on the degree of helix kink. To see this figure in color, go online.

Figure 3

OmpA monomer interactions with PGN. (A) A snapshot depicts the end of one of the simulations of the OmpA monomer (blue) in the presence of BLP (green), highlighting the interactions of the CTD with the PGN network (red). Given here are residues involved in PGN contacts illustrated in van der Waals representation. (B) Shown here is the minimum distance between OmpA CTD and the PGN network for all four simulations of OmpA monomer with BLP. (C) Given here is the distance between the PGN network and the OM measured along the z axis between the centers of geometry of the PGN sugar strands and the phosphorus atoms on the lower leaflet of the OM. This is averaged over all four independent simulations and the error bars indicate SDs. To see this figure in color, go online.

Figure 4

OmpA monomer interactions with the OM. (A) A snapshot depicts the end of one of the simulations of the OmpA monomer (blue) without BLP, highlighting the interactions of the CTD and the membrane (gray spheres represents the phosphorus atoms). (B) Minimum distance is given between OmpA CTD and the PGN network for all four simulations of OmpA monomer without BLP. (C) Distance between the PGN network and the OM is measured as described in Fig. 3, averaged over all four independent simulations. Error bars indicate SDs. To see this figure in color, go online.

Figure 5

OmpA dimer interactions with PGN. (A) Shown here is the length of the unstructured linker connecting OmpA NTD and CTD (blue), plotted against the distance between the PGN network and the OM (red) for one of the simulations of OmpA dimer without BLP. The former is measured as previously described (28), whereas the latter is measured as in Fig. 3. (B) Given here are snapshots of this simulation at three different time points, highlighting the interactions between OmpA CTD (blue) and the PGN network (red). (C) The z coordinates of the PGN network are projected into a surface representation at these three time points to illustrate undulations observed during the simulations. The “x” indicates a local buckling effect induced by interactions with OmpA CTD. To see this figure in color, go online.

Figure 6

OmpA interactions with BLP. (A) A snapshot depicts the end of one of the simulations of OmpA dimer (blue) with BLP (green), highlighting their interactions. Enlarged image shows residues involved in these interactions from both proteins. (B) Minimum distance between the BLP helices and the OmpA CTD for all simulations of OmpA dimer is shown in blue, whereas the minimum distance between the BLP lipid tails and the OmpA NTD is shown in red dashed lines. (C) Electrostatic profile of the BLP is calculated using APBS (55) in PyMOL (18). (D) Shown here is contact analysis performed for each residue of the BLP averaged over all simulations where OmpA-BLP interactions were observed. A score of 1 indicates contacts throughout the entire 100-ns simulation. A distance cutoff of 4 Å was used for this analysis. To see this figure in color, go online.