By: Michelle Rudden
Highly conserved domains in MCE proteins are used as structural building block to synthesise diverse transport architectures capable of shuttling hydrophobic lipid molecules between the inner and outer membrane of bacteria.
One of the defining hallmarks of Gram negative bacteria is their cell wall, composed of two phospholipid (PL) bilayer membranes separated by the periplasmic space which contains a thin layer of peptidoglycan. The inner membrane (IM) faces the cytoplasm while the outer membrane (OM) is exposed to the environment. The physical properties of the OM is a crucial selective barrier often preventing entry of toxic compounds, antibiotics and host innate immune compounds such as cationic antimicrobial peptides (CAMPs). Any mutations or changes affecting the physical integrity of the OM can reduce pathogenicity and increase antibiotic susceptibly, as a result it is a therapeutic target for many bacterial diseases. The OM is an asymmetric bilayer composed of an inner leaflet of phospholipids and an outer leaflet rich in lipopolysaccharide (LPS) in which several non-specific porins and specific uptake channels are embedded. The LPS-bilayer structure of the OM is more rigid compared to normal phospholipid bilayers and is largely impermeable to hydrophobic molecules by slowing down passive diffusion and narrow pores limit by size the uptake of hydrophilic molecules. The transport system for trafficking LPS molecules from the cytoplasm has been described in detail with a model comprising seven essential proteins (with most of the structures resolved) that spans the periplasmic space to deliver the LPS molecule to the outer leaflet of the OM. However there is no detailed mechanism described for the transport of nascent phospholipids in the inner leaflet and exactly how the asymmetry is maintained between the leaflets of the OM still remains unclear.
In a paper published this month in Cell, Ekiert et al, (2017) propose a mechanism by which proteins belonging to the mammalian cell entry (MCE) superfamily form hexameric assemblies that can form diverse protein channels with hydrophobic pores capable of shuttling hydrophobic molecules between the IM and OM in E. coli. MCE proteins are ubiquitous in double membraned bacteria including E. coli and some membrane bound organelles such as chloroplasts where they are suggested to be involved in transport of lipid molecules either from the OM to the IM or as exporters driving molecules out of the cell.
Lipid molecules are hydrophobic and are poorly soluble in water, the hydrophilic periplasmic space is the major barrier in lipid transport across the membranes. In the LPS transport model protein components assemble to form a periplasm spanning bridge with a hydrophobic core that protects the lipid tails of LPS facilitating the movement of LPS molecules toward the OM. This led Ekiert et al (2017) to propose a similar mechanism by which MCE proteins assemble to form a hydrophobic pore allowing the transport of phospholipids between inner and outer membranes.
In this paper X-Ray and Cryo-electron microscopy (Cryo-EM) structures are reported for three MCE proteins found in E. coli each producing a distinct architecture with conserved MCE hexameric ring modules. The crystal structure of the MCE protein MlaD in E. coli revealed a single homo-hexameric ring assembled with a hydrophobic core that facilitates the transport of phospholipids. MlaD forms a complex with three other proteins anchored in the IM (Fig 1). MlaD does not span the periplasmic space, it recruits periplasmic lipid binding protein MlaC that delivers the phospholipids to the outer membrane.
Cryo EM structures of two different MCE proteins non-homologous to MlaD revealed different architectures that span the periplasm implicating their role in moving hydrophobic substrates across the membrane. PqiB forms a needle and syringe like structure that forms a periplasmic bridge (Fig 1). PqiB contains three MCE domains (MCE1, MCE2, MCE3) each forming a hexameric ring stacked on top of each other to form the hollow barrel of the structure (Fig. 1). The needle like structure of PqiB is a six helix coiled coil, one helix contributed from the C-terminus of each polypeptide chain. The needle contains a hollow hydrophobic lumen that runs through the barrel thus allowing for the movement of lipid molecules across the periplasm. The YebT MCE protein also forms a periplasmic bridge with a unique structure. YebT contains seven MCE domains that stack on top of each other as hexameric rings forming an elongated tube, like both MlaD and PqiB, YebT is anchored in the IM by N-terminal trans membrane (TM) helices. The elongated tube also contains a hydrophobic lumen which allow the transport of hydrophobic substrates. Both YebT and PqiB are greater than 200 Å in length which is approximately the width of the periplasm. Ekiert et al., (2017) propose that YebT and PqiB provide a continuous pathway for lipid translocation across membranes without the need for a soluble periplasmic binding protein in a manner similar to that of the current LPS model.
Ekiert and colleagues’ present a neat structural argument for role of MCE domains in generating diverse architectures that are capable of spanning the periplasm and may function to traffic lipid across membranes. However the authors do state that there is still “a great deal that we don’t understand about the components of MCE transporters and how they function together”. This leads to several exciting open questions like do these systems function together or alone and are all three needed in E. coli? What is the directionality of lipid transport? What are the substrates for these systems? How phospholipids are transported and inserted in the outer membrane could be essential to understand how assembly and asymmetry is maintained in the OM.
Source: Ekiert et al., (2017) Architectures of Lipid Transport Systems for the Bacterial Outer Membrane. Cell 169, 273–285