The architecture of the OmpC–MlaA complex sheds light on the maintenance of outer membrane lipid asymmetry in Escherichia coli

Jiang Yeow,Kang Wei Tan,Daniel A Holdbrook,Zhi-Soon Chong,Jan K Marzinek,Peter J Bond,Shu-Sin Chng

doi:10.1074/jbc.ra118.002441

Abstract

A distinctive feature of the Gram-negative bacterial cell envelope is the asymmetric outer membrane (OM), where lipopolysaccharides and phospholipids (PLs) reside in the outer and inner leaflets, respectively. This unique lipid asymmetry renders the OM impermeable to external insults, including antibiotics and bile salts. In Escherichia coli, the complex comprising osmoporin OmpC and the OM lipoprotein MlaA is believed to maintain lipid asymmetry by removing mislocalized PLs from the outer leaflet of the OM. How this complex performs this function is unknown. Here, we defined the molecular architecture of the OmpC-MlaA complex to gain insights into its role in PL transport. Using in vivo photo-cross-linking and molecular dynamics simulations, we established that MlaA interacts extensively with OmpC and is located entirely within the lipid bilayer. In addition, MlaA forms a hydrophilic channel, likely enabling PL translocation across the OM. We further showed that flexibility in a hairpin loop adjacent to the channel is critical in modulating MlaA activity. Finally, we demonstrated that OmpC plays a functional role in maintaining OM lipid asymmetry together with MlaA. Our work offers glimpses into how the OmpC-MlaA complex transports PLs across the OM and has important implications for future antibacterial drug development.

Highlights

A distinctive feature of the Gram-negative bacterial cell envelope is the asymmetric outer membrane (OM), where lipopolysaccharides and phospholipids (PLs) reside in the outer and inner leaflets, respectively
This function is fully dependent on the establishment and maintenance of lipid asymmetry; cells generally become more sensitive to external insults when OM lipid asymmetry is disrupted, which is typically characterized by the accumulation of PLs in the outer leaflet [4, 5]
We have previously proposed that OmpC may allow MlaA to traverse the bilayer and gain access to PLs that have accumulated in the outer leaflet of the OM [12]

Summary

Results

The OmpC trimer contacts MlaA directly along its membranefacing dimeric interfaces To develop a detailed architectural understanding of the OmpC–MlaA complex, we carried out in vivo photo-crosslinking to map intermolecular interactions within the complex. The positions of the two OmpC-contacting peptides on the MlaA model are spatially separated in a way consistent with the arrangement of the residues on OmpC that cross-link to each peptide (Fig. 2D) This reveals how MlaA may potentially be oriented and organized around the dimeric interface of the OmpC trimer and suggests that the entire MlaA molecule may reside in the membrane. These structures revealed that MlaA interacts with trimeric porins in the membrane at one or more of their dimeric interfaces (Fig. S11B), in an orientation similar to one of our simulated OmpC–MlaA models (Fig. 3B) They showed that MlaA contains a hydrophilic channel. We conclude that OmpC has a functional role in maintaining OM lipid symmetry together with MlaA

Discussion

Experimental procedures

Simulation procedures and setup