LetB Structure Reveals a Tunnel for Lipid Transport across the Bacterial Envelope

Isom, Georgia L.; Coudray, Nicolas; MacRae, M.R.; McManus, C.T.; Ekiert, Damian; Bhabha, Gira

doi:10.1016/j.cell.2020.03.030

Cited by 49 publications

(80 citation statements)

References 88 publications

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“…MlaD forms a hexameric MCE ring with a hydrophobic pore at the center ( Ekiert et al, 2017 ). Similar hydrophobic tunnels have been observed through the MCE rings of PqiB and LetB ( Ekiert et al, 2017 ; Isom et al, 2020 ), and phospholipids have been cross-linked inside the tunnel of LetB. Indeed, in our structure of the MlaFEDB complex, the pore of MlaD and the outward-open pocket of MlaE line up with each other, resulting in a continuous hydrophobic pathway that runs from the pocket of MlaE, through MlaD, and out into the periplasm ( Figure 4B ).…”

Section: Resultssupporting

confidence: 76%

“…To assess whether phospholipids are bound in the pocket of MlaE in cells, we utilized a site-specific photocross-linking method ( Isom et al, 2020 ) to detect binding of radiolabeled phospholipids in vivo. We incorporated the unnatural photocross-linking amino acid p -benzoyl-L-phenylalanine (BPA) ( Chin et al, 2002 ) into the MlaE protein at five positions in the lipid-binding site (Leu70, Val77, Leu78, Tyr81, and Leu99), as well as Phe209 (protected in the MlaE core; not expected to contact lipids) or Trp149 (membrane exposed; expected to contact bulk membrane lipids) ( Figure 5A ).…”

Section: Resultsmentioning

confidence: 99%

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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: Resultssupporting

confidence: 76%

Section: Resultsmentioning

confidence: 99%

Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

Coudray

Isom

MacRae

et al. 2020

eLife

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“…MlaD forms a hexameric MCE ring with a hydrophobic pore at the center (Ekiert et al, 2017). Similar hydrophobic tunnels have been observed through the MCE rings of PqiB and LetB (Ekiert et al, 2017;Isom et al, 2020), and phospholipids have outward-open pocket formed by TM1 (salmon helices) and TM2 (orange helices). The boundary of the substrate-binding pocket was estimated using CASTp (Tian et al, 2018), and is displayed as a red surface.…”

Section: Mlae Adopts An Outward-open Conformation In the Apo Statesupporting

confidence: 57%

“…To assess whether phospholipids are bound in the pocket of MlaE in cells, we utilized a sitespecific photocrosslinking method (Isom et al, 2020) to detect binding of radiolabeled phospholipids in vivo.…”

Section: Lipids Are Bound In the Substrate-translocation Pathwaymentioning

confidence: 99%

Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

Coudray

Isom

MacRae

et al. 2020

Preprint

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In double-membraned bacteria, phospholipids must be transported across the cell envelope to maintain the outer membrane barrier, which plays a key role in antibiotic resistance and pathogen virulence. The Mla pathway has been implicated in phospholipid trafficking and outer membrane integrity, but the mechanism by which this ABC transporter facilitates phospholipid transport remains unknown. Here we report the cryo-EM structure of the MlaFEDB inner membrane complex at 3.05 Å resolution. Our structure reveals that the core transporter subunit, MlaE, adopts an ABC exporter fold and is related to the lipopolysaccharide export system as well as the human lipid exporter families, ABCA and ABCG. Unexpectedly, two phospholipids are bound in the outward-facing pocket of MlaFEDB, raising the possibility that multiple lipid substrates may be translocated each transport cycle. The unusual configuration of bound lipids suggests that lipid reorientation may occur before extrusion through the hydrophobic MlaD pore. Site-specific crosslinking confirms that lipids bind in this pocket in vivo. Our structure supports a model in which the Mla system may drive phospholipid export to the bacterial outer membrane, and provides mechanistic insight into the function of an exporter family conserved from bacteria to humans.

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“…Additionally, the outer membrane must be constricted in conjunction with the peptidoglycan cell wall during division [13]. To achieve this, Gram-negative bacteria have evolved a network of interlinked pathways for the construction and maintenance of the outer membrane [12,[14][15][16][17][18][19][20][21][22]. Despite considerable progress in understanding how these pathways function in E. coli, in many cases, the proteins that constitute them are not well characterized, and additional pathways likely remain to be identified [12,20,23].…”

Section: Introductionmentioning

confidence: 99%

BonA fromAcinetobacter baumanniiforms a divisome-localized decamer that supports outer envelope function

Grinter

Morris

Dunstan

et al. 2020

Preprint

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Acinetobacter baumannii is a high-risk pathogen due to the rapid global spread of multi-drug resistant lineages. Its phylogenetic divergence from other ESKAPE pathogens means that determinants of its antimicrobial resistance can be difficult to extrapolate from other widely studied bacteria. A recent study showed that A. baumannii upregulates production of an outer-membrane lipoprotein, which we designate BonA, in response to challenge with polymyxins. Here we show that BonA has limited sequence similarity and distinct structural features compared to lipoproteins from other bacterial species. Analyses through X-ray crystallography, small-angle X-ray scattering, electron microscopy, and multiangle light scattering demonstrate that BonA has a dual BON-domain architecture and forms a decamer via an unusual oligomerization mechanism. This analysis also indicates this decamer is transient, suggesting dynamic oligomerization plays a role in BonA function. Antisera recognizing BonA shows it is an outer membrane protein localized to the divisome. Loss of BonA modulates the density of the outer membrane, consistent with a change in its structure or link to the peptidoglycan, and prevents motility in a clinical strain (ATCC 17978). Consistent with these findings, the dimensions of the BonA decamer are sufficient to permeate the peptidoglycan layer, with the potential to form a membrane-spanning complex during cell division.

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LetB Structure Reveals a Tunnel for Lipid Transport across the Bacterial Envelope

Abstract: Highlights d Cryo-EM structures of LetB reveal a tunnel that spans the bacterial cell envelope d Lipids bind inside the tunnel, supporting a role for LetB in lipid transport d Different conformations reveal tunnel dynamics, with implications for transport

Cited by 49 publications

References 88 publications

Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

BonA fromAcinetobacter baumanniiforms a divisome-localized decamer that supports outer envelope function

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