3.A.1.27.3
ABC transporter maintaining outer membrane (OM) lipid asymmetry, MlaABCDEF (YrbABCDEF) (Malinverni and Silhavy, 2009). MlaA (VacJ) is a "spreading" protein, essential for Shigella pathogenicity (Suzuki et al., 1994).  The ABC transporter, MlaEFBD, provides energy for maintaining OM lipid asymmetry via the transport of aberrantly localized phospholipids (PLs) from the OM to the inner membrane (IM) (Thong et al. 2016). MlaD spans the periplasm, forms stable hexamers within the complex, functions in substrate binding with strong affinity for PLs within a channel that spans the periplasm, and modulates ATP hydrolytic activity. MlaB plays critical roles in both the assembly and activity of the transporter.  MlaA forms a complex with OmpC and OmpF in the outer membrane to extract PLs from the outer leaflet of the OM (Chong et al. 2015). MlaA is a monomeric 2 α-helical TMS OM protein that functions as a phospholipid translocation channel, forming a ~20-Å-thick doughnut embedded in the inner leaflet of the OM with a central, amphipathic pore (Abellón-Ruiz et al. 2017). This architecture prevents access of inner leaflet phospholipids to the pore, but allows outer leaflet phospholipids to bind to a pronounced ridge surrounding the channel. Members of the mammalian cell entry (MCE) protein family, one of which is MlaD, form hexameric assemblies with a central channel capable of mediating lipid transport across the periplasm (Ekiert et al. 2017). MlaD forms a ring associated with the ABC transporter complex in the inner membrane. A soluble lipid-binding protein, MlaC ferries lipids between MlaD and an outer membrane protein complex. EM structures of two other E. coli MCE proteins show that YebT (LetB) forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the periplasmic space. These homologs transport lipids between the two membranes of Gram-negative bacteria, some eukaryotic organelles and possibly actinobacteria (Ekiert et al. 2017). MCE systems mediate phospholipid trafficking across the periplasm. ~3.5 Å cryo-EM structures of the E. coli MCE protein LetB reveals an ~0.6 megadalton complex that consists of seven stacked rings, with a central hydrophobic tunnel sufficiently long to span the periplasm (Isom et al. 2020). Lipids bind inside the tunnel, suggesting that it functions as the pathway for lipid transport. Cryo-EM structures in the open and closed states revealed a dynamic tunnel lining with implications for gating and substrate translocation. These results support a model in which LetB establishes a physical link between the two membranes and creates a hydrophobic pathway for the translocation of lipids across the periplasm (Isom et al. 2020). The transmembrane subunit, MlaE, has minimal sequence similarity to other transporters. Coudray et al. 2020 reported the cryo-EM structure of MlaFEDB at 3.05 Å resolution, revealing distant relationships to the LPS and MacAB transporters, as well as members of the eukaryotic ABCA/ABCG families. A continuous transport pathway extends from the MlaE substrate- binding site, through the channel of MlaD, and into the periplasm. Two phospholipids are bound to MlaFEDB, suggesting that multiple lipid substrates may be transported each cycle. The structure provides mechanistic insight into substrate recognition and transport by MlaFEDB (Coudray et al. 2020). Structures of both the E. coli and P. aeruginosa MlaFEDB complexes have been determined by cryoEM (Zhou et al. 2021). The structures show that the MlaFEBD complex contains a total of twelve protein molecules with a stoichiometry of MlaF₂E₂B₂D₆, and binds a plethora of phospholipids (PLs) at different locations. In contrast to canonical ABC transporters, nucleotide binding fails to trigger significant conformational changes of both MlaFEBD and MlaFEB in the nucleotide-binding and transmembrane domains, correlated with their low ATPase activities exhibited in both detergent micelles and lipid nanodiscs. PLs or detergents appeared to relocate to the membrane-proximal end from the distal end of the hydrophobic tunnel formed by the MlaD hexamer in MlaFEBD upon addition of ATP, indicating that retrograde PL transport might occur in the tunnel in an ATP-dependent manner. Site-specific photocrosslinking experiment confirmed that the substrate-binding pocket in the dimeric MlaE and the MlaD hexamer are able to bind PLs in vitro, in line with the notion that the MlaFEBD complex functions as a PL transporter (Zhou et al. 2021).  Mla uses a shuttle-like mechanism to move lipids between the MlaFEDB inner membrane complex and the MlaA-OmpF/C OM complex, via a periplasmic lipid-binding protein, MlaC. MlaC binds to MlaD and MlaA (MacRae et al. 2023). They mapped the MlaC-MlaA and MlaC-MlaD protein-protein interfaces, suggesting that the MlaD and MlaA binding surfaces on MlaC overlap to a large extent, leading to a model in which MlaC can only bind one of these proteins at a time. Low-resolution cryo-EM maps of MlaC bound to MlaFEDB suggested that at least two MlaC molecules can bind to MlaD at once, in a conformation consistent with AlphaFold2 predictions (MacRae et al. 2023). The Mla system of the diderm Firmicute, Veillonella parvula, reveals an ancestral transenvelope bridge for phospholipid trafficking (Grasekamp et al. 2023). RamA upregulates mlaFEDCB to mediate resistance to tetracycline-class antibiotics and the stability of membranes in Klebsiella pneumoniae (Zhao et al. 2024).  This transporter may be part of a nanomachine (Bilsing et al. 2023).