Structure and lipid dynamics in theA. baumanniimaintenance of lipid asymmetry (MLA) inner membrane complex

Abstract

24The maintenance of lipid asymmetry (MLA) system is involved in lipid transport from/to 25 the outer membrane in gram-negative bacteria, and contributes to broad-range 26 antibiotic resistance. Here, we report the cryo-EM structure of the A. baumannii 27 MlaBDEF core complex, in the apo, ADP-and AppNHp-bound states. This reveals 28 multiple lipid binding sites, and suggests a mechanism for their transport. 29 30 31Gram-negative bacteria are enveloped by two lipid bilayers, separated by the periplasmic 32 space containing the peptidoglycan cell wall. The two membranes have distinct lipid 33 composition: The inner membrane (IM) consists of glycerophospholipids, with both leaflets 34 having similar compositions, while the outer membrane (OM) is asymmetric, with an outer 35 leaflet of lipopolysaccharides (LPS) and an inner leaflet of glycerophospholipids (1). This lipid 36 gradient is maintained by several machineries, including YebT, PqiB, and the multicomponent 37MLA system (2, 3), which consists of MlaA present in the OM (4, 5), the shuttle MlaC in the 38 periplasmic space, and the MlaBDEF ABC transporter system in the IM (6). The directionality 39 of lipid transport by the MLA system has been the subject of debate, with initial reports 40 suggesting that it recycles lipids from the OM to the IM (7), but recent results (6, 8) indicated 41 that it might exports glycerophospholipids to the outer membrane. Low-resolution cryo-EM 42 maps of the MlaBDEF core complex, from Escherichia coli (2) and Acinetobacter baumannii 43 (6) have revealed the overall architecture of the complex, but did not allow to elucidate the 44 molecular details of lipid binding and transport. 45To address the mechanism of MLA functioning, we have determined the cryo-EM structure of 46 the A. baumannii MlaBDEF complex, bound to the non-hydrolizable ATP analogue AppNHp, 47

Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

“…On the other hand, prior studies indicated that the Mla system is an importer (Malinverni and Silhavy, 2009;Chong, Woo and Chng, 2015;Powers and Trent, 2018;Yeow et al, 2018), and in vitro reconstitution and transport assays suggest that MlaFEDB may be bi-directional, with a preference for import (retrograde transport) (Tang et al, 2020). Concurrently with our preprint, two other groups also reported structures of the MlaFEDB complex on BioRxiv (Coudray et al, 2020;Mann et al, 2020;Tang et al, 2020), and these structures appear to be similar overall, although the PDB coordinates…”

Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

“…It is noteworthy that the proposed manner of lipid binding to MlaFEDB differs significantly among all three pre-prints posted around the same time. Our structure and the other E. coli MlaFEDB structure described by Tang, et al describe lipid binding to the outward-open pocket of MlaE (Tang et al, 2020), while the Acinetobacter baumannii MlaFEDB described by Mann, et al describes lipid binding sites at the pore of MlaD as well as six additional lipidbinding sites in between the pore loops of each of the six MCE domains in the MlaD ring (Mann et al, 2020). Tang et al observed density assigned to a single phospholipid bound in the outward-open pocket of MlaFEDB, with the head group facing towards the core of the MlaE dimer and tails pointing towards the MlaD pore.…”

Structural and functional diversity calls for a new classification of ABC transporters

“…Of note, at the time of writing, publicly available, yet unpublished structures of the lipid transporter complex MlaFEDB of Gram ‐negative bacteria reveal some resemblance of MlaE to LptF/G and MacB. However, the number of TM helices differs between LptFG (six TM helices), MlaE (five TM helices), and MacB (four TM helices) [45–48] (Table S6 and Fig. S1).…”