In Gram-negative bacteria, lipid asymmetry is critical for the function of the outer membrane (OM) as a selective permeability barrier, but how it is established and maintained is poorly understood. Here, we characterize a non-canonical ATP-binding cassette (ABC) transporter in Escherichia coli that provides energy for maintaining OM lipid asymmetry via the transport of aberrantly localized phospholipids (PLs) from the OM to the inner membrane (IM). We establish that the transporter comprises canonical components, MlaF and MlaE, and auxiliary proteins, MlaD and MlaB, of previously unknown functions. We further demonstrate that MlaD forms extremely stable hexamers within the complex, functions in substrate binding with strong affinity for PLs, and modulates ATP hydrolytic activity. In addition, MlaB plays critical roles in both the assembly and activity of the transporter. Our work provides mechanistic insights into how the MlaFEDB complex participates in ensuring active retrograde PL transport to maintain OM lipid asymmetry.DOI: http://dx.doi.org/10.7554/eLife.19042.001

The three-dimensional structure of nawaprin has been determined by nuclear magnetic resonance spectroscopy. This 51-amino acid residue peptide was isolated from the venom of the spitting cobra, Naja nigricollis, and is the first member of a new family of snake venom proteins referred to as waprins. Nawaprin is relatively flat and disc-like in shape, characterized by a spiral backbone configuration that forms outer and inner circular segments. The two circular segments are held together by four disulfide bonds, three of which are clustered at the base of the molecule. The inner segment contains a short antiparallel ␤-sheet, whereas the outer segment is devoid of secondary structures except for a small turn or 3 10 helix. The structure of nawaprin is very similar to elafin, a human leukocyte elastase-specific inhibitor. Although substantial parts of the nawaprin molecule are well defined, the tips of the outer and inner circular segments, which are hypothesized to be critical for binding interactions, are apparently disordered, similar to that found in elafin. The amino acid residues in these important regions in nawaprin are different from those in elafin, suggesting that nawaprin is not an elastase-specific inhibitor and therefore has a different function in the snake venom.Snake venoms are rich sources of pharmacologically active polypeptides and proteins. Some of these proteins exhibit enzymatic activities. These enzymes include phospholipase A 2 , proteinase, nucleotidase, phosphodiesterase, and L-amino acid oxidase. In addition to their catalytic properties that may contribute to the digestive action of the venom, these enzymes also induce various pharmacological effects including neurotoxic, myotoxic, cardiotoxic, hemorrhagic, hemolytic, procoagulant, and anticoagulant effects (1, 2). Several other snake venom proteins and polypeptides do not exhibit these and other enzymatic activities and thus are described as "nonenzymatic proteins." These proteins include neurotoxins, cardiotoxins, myotoxins, ion channel inhibitors, and anticoagulant proteins (3, 4). Thus, snake venom proteins, whether they are enzymatic or nonenzymatic, have evolved as a complex mixture of proteins that target several tissues, organs, and physiological systems and interfere in their normal functions. Therefore snake venoms, when injected into a prey or victim, result in the simultaneous assault on various tissues, leading to multiple organ or system failure and often death.A large number of protein toxins have been purified and characterized from snake venoms. These studies have shown that each venom contains over a hundred protein toxins. These toxins, however, belong to a very small number of superfamilies of proteins. For example, a single snake venom can contain as many as 15 isoforms of phospholipase A 2 (5-7). As one would expect, they share remarkable similarities in their primary, secondary, and tertiary structures. However, at times they differ from each other in their biological targeting and hence their pharmacological effec...

In Gram-negative bacteria, lipid asymmetry is critical for the function of the outer membrane (OM) as a selective permeability barrier, but how it is established and maintained is poorly understood. Here, we characterize a non-canonical ATP-binding cassette (ABC) transporter in Escherichia coli that provides energy for maintaining OM lipid asymmetry via the transport of aberrantly localized phospholipids (PLs) from the OM to the inner membrane (IM). We establish that the transporter comprises canonical components, MlaF and MlaE, and auxiliary proteins, MlaD and MlaB, of previously unknown functions. We further demonstrate that MlaD forms extremely stable hexamers within the complex, functions in substrate binding with strong affinity for PLs, and modulates ATP hydrolytic activity. In addition, MlaB plays critical roles in both the assembly and activity of the transporter. Our work provides mechanistic insights into how the MlaFEDB complex participates in ensuring active retrograde PL transport to maintain OM lipid asymmetry.