Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

Coudray, Nicolas; Isom, Georgia L.; MacRae, M.R.; Saiduddin, Mariyah N.; Bhabha, Gira; Ekiert, D.C.

doi:10.1101/2020.06.02.129247

Cited by 11 publications

(24 citation statements)

References 73 publications

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“…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…”

Section: Discussionsupporting

confidence: 71%

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Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

Coudray

Isom

MacRae

et al. 2020

eLife

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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.

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Section: Discussionsupporting

confidence: 71%

“…Three replicates of the experiment were performed, starting with protein expression. NB: earlier protocols using urea solubilization (Coudray et al, 2020) gave globally similar results but with variation in cross linking efficiency between biological replicates; the improved protocol used here,…”

Section: Lipid Crosslinking Experimentsmentioning

confidence: 72%

Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

Coudray

Isom

MacRae

et al. 2020

eLife

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“…While this manuscript was under review, three independent groups released the structure of the Escherichia coli MlaBDEF complex (MlaBDEFec), in a range of nucleotide states and with various solubilization approaches (18)(19)(20).…”

Section: Discussionmentioning

confidence: 99%

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

Mann

Fan

Farrell

et al. 2020

Preprint

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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

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“…A spectacular high resolution example is provided by the cryo‐EM structure of the phospholipid ABC transporter, MlaFEDB, in Nanodics formed with MSP1D1 and an E. coli polar lipid extract (PDB http://firstglance.jmol.org/fg.htm?mol=6XBD) that explicitly resolves the encircling MSP (Figure 3a). 112 This transporter was found with two lipid molecules bound as substrates, which may indicate transport of two lipids in each catalytic cycle, or involvement of this transporter in the transport of cardiolipin, which has four acyl chains.…”

Section: Introductionmentioning

confidence: 94%

“…(a) Cryo‐electron microscopy (cryo‐EM) structure of the ABC transporter complex MlaFEDB. Hexamer MLAD (yellow), dimeric transport permease MLAE (red), ATP binding protein (cyan) and transporter‐binding protein MLAB (blue) are enclosed into MSP1D1 Nanodisc (two parallel helices shown in green) 112 . (b) Cryo‐EM structure of connexin‐46/50 incorporated in two Nanodiscs (light gray contours).…”

Section: Introductionmentioning

confidence: 99%

Nanodiscs: A toolkit for membrane protein science

2020

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Membrane proteins are involved in numerous vital biological processes, including transport, signal transduction and the enzymes in a variety of metabolic pathways. Integral membrane proteins account for up to 30% of the human proteome and they make up more than half of all currently marketed therapeutic targets. Unfortunately, membrane proteins are inherently recalcitrant to study using the normal toolkit available to scientists, and one is most often left with the challenge of finding inhibitors, activators and specific antibodies using a denatured or detergent solubilized aggregate. The Nanodisc platform circumvents these challenges by providing a self‐assembled system that renders typically insoluble, yet biologically and pharmacologically significant, targets such as receptors, transporters, enzymes, and viral antigens soluble in aqueous media in a native‐like bilayer environment that maintain a target's functional activity. By providing a bilayer surface of defined composition and structure, Nanodiscs have found great utility in the study of cellular signaling complexes that assemble on a membrane surface. Nanodiscs provide a nanometer scale vehicle for the in vivo delivery of amphipathic drugs, therapeutic lipids, tethered nucleic acids, imaging agents and active protein complexes. This means for generating nanoscale lipid bilayers has spawned the successful use of numerous other polymer and peptide amphipathic systems. This review, in celebration of the Anfinsen Award, summarizes some recent results and provides an inroad into the current and historical literature.

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Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

Cited by 11 publications

References 73 publications

Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

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

Nanodiscs: A toolkit for membrane protein science

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