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In double-membraned bacteria, phospholipid transport across the cell envelope is critical to maintain the outer membrane barrier, which plays a key role in virulence and antibiotic resistance. An MCE transport system called Mla has been implicated in phospholipid trafficking and outer membrane integrity, and includes an ABC transporter, MlaFEDB. The transmembrane subunit, MlaE, has minimal sequence similarity to other transporters, and the structure of the entire inner-membrane MlaFEDB complex remains unknown. Here, we report the cryo-EM structure of MlaFEDB at 3.05 Å resolution, revealing distant relationships to the LPS and MacAB transporters, as well as 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. Unexpectedly, two phospholipids are bound to MlaFEDB, suggesting that multiple lipid substrates may be transported each cycle. Our structure provides mechanistic insight into substrate recognition and transport by MlaFEDB., Competing Interests: NC, GI, MM, MS, GB, DE No competing interests declared, (© 2020, Coudray et al.)

ABC transporters facilitate the movement of diverse molecules across cellular membranes, but how their activity is regulated post-translationally is not well understood. Here we report the crystal structure of MlaFB from E. coli , the cytoplasmic portion of the larger MlaFEDB ABC transporter complex, which drives phospholipid trafficking across the bacterial envelope to maintain outer membrane integrity. MlaB, a STAS domain protein, binds the ABC nucleotide binding domain, MlaF, and is required for its stability. Our structure also implicates a unique C-terminal tail of MlaF in self-dimerization. Both the C-terminal tail of MlaF and the interaction with MlaB are required for the proper assembly of the MlaFEDB complex and its function in cells. This work leads to a new model for how an important bacterial lipid transporter may be regulated by small proteins, and raises the possibility that similar regulatory mechanisms may exist more broadly across the ABC transporter family., Competing Interests: LK, YC, NC, SG, MM, GI, ET, GB, DE No competing interests declared, (© 2020, Kolich et al.)