Suppressor Mutations in LptF Bypass Essentiality of LptC by Forming a Six-Protein Transenvelope Bridge That Efficiently Transports Lipopolysaccharide

Falchi, Federica A.; Taylor, Rebecca J.; Rowe, Sebastian J; Moura, Elisabete C C M; Baeta, Tiago; Laguri, Cédric; Simorre, Jean-Pierre; Kahne, Daniel; Polissi, Alessandra; Sperandeo, Paola

doi:10.1128/mbio.02202-22

Cited by 6 publications

(2 citation statements)

References 41 publications

(104 reference statements)

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“…The LILBID-MS data show that LPS is released upon ATP hydrolysis by LptB 2 FG ( Figure 6H-J, Figure 7 steps 1-2 ). Thus, LptC is required for a productive transport cycle in the wild-type protein 24,57,58 . Our observations suggest that the TM-LptC dynamically associates and dissociates at the lateral gate-2, even in the apo-state while its β-jellyroll domain is firmly bound to LptF β-jellyroll ( Figure 7, step 3 ).…”

Section: Discussionmentioning

confidence: 99%

Dynamic basis of lipopolysaccharide export by LptB₂FGC

Dajka,

Rath,

Morgner

et al. 2024

Preprint

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Lipopolysaccharides (LPS) confer resistance against harsh conditions, including antibiotics, in Gram-negative bacteria. The lipopolysaccharide transport (Lpt) complex, consisting of seven proteins (A-G), exports LPS across the cellular envelope. LptB2FG forms an ATP-binding cassette transporter that transfers LPS to LptC. How LptB2FG couples ATP binding and hydrolysis with LPS transport to LptC remains unclear. We observed the conformational heterogeneity of LptB2FG and LptB2FGC in micelles and/or proteoliposomes using pulsed dipolar electron spin resonance spectroscopy. Additionally, we monitored LPS binding and release using laser-induced liquid bead ion desorption mass spectrometry. The β-jellyroll domain of LptF stably interacts with the LptG and LptC β-jellyrolls in both the apo and vanadate-trapped states. ATP binding at the cytoplasmic side is allosterically coupled to the selective opening of the periplasmic LptF β-jellyroll domain. In LptB2FG, ATP binding closes the nucleotide binding domains, causing a collapse of the first lateral gate as observed in structures. However, the second lateral gate, which forms the putative entry site for LPS, exhibits a heterogeneous conformation. LptC binding limits the flexibility of this gate to two conformations, likely representing the helix of LptC as either released from or inserted into the transmembrane domains. Our results reveal the regulation of the LPS entry gate through the dynamic behavior of the LptC transmembrane helix, while its β-jellyroll domain is anchored in the periplasm. This, combined with long-range ATP-dependent allosteric gating of the LptF β-jellyroll domain, may ensure efficient and unidirectional transport of LPS across the periplasm.

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Section: Discussionmentioning

confidence: 99%

Dynamic basis of lipopolysaccharide export by LptB₂FGC

Dajka,

Rath,

Morgner

et al. 2024

Preprint

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“…Therefore, the helix might reduce futile ATP hydrolysis by coupling it efficiently to substrate extraction [ 78 , 79 , 81 ]. Lethal LptC deletions can be suppressed when the arginine residue R212 in the β-jellyroll domain of LptF is substituted with glycine, restoring wildtype LPS transport and ATPase activity of the transporter, even though affinity to LptA is reduced [ 103 , 104 ]. Interestingly, this point mutation is still able to form a complex with LptC when it is present.…”

Section: The Lpt Systemmentioning

confidence: 99%

ABC Transporters in Bacterial Nanomachineries

Bilsing

Anlauf

Hachani

et al. 2023

IJMS

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Members of the superfamily of ABC transporters are found in all domains of life. Most of these primary active transporters act as isolated entities and export or import their substrates in an ATP-dependent manner across biological membranes. However, some ABC transporters are also part of larger protein complexes, so-called nanomachineries that catalyze the vectorial transport of their substrates. Here, we will focus on four bacterial examples of such nanomachineries: the Mac system providing drug resistance, the Lpt system catalyzing vectorial LPS transport, the Mla system responsible for phospholipid transport, and the Lol system, which is required for lipoprotein transport to the outer membrane of Gram-negative bacteria. For all four systems, we tried to summarize the existing data and provide a structure-function analysis highlighting the mechanistical aspect of the coupling of ATP hydrolysis to substrate translocation.

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Conformational Investigation of the Asymmetric Periplasmic Domains of E. coli LptB2FGC Using SDSL CW EPR Spectroscopy

Cina

Klug

2023

Appl Magn Reson

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Suppressor Mutations in LptF Bypass Essentiality of LptC by Forming a Six-Protein Transenvelope Bridge That Efficiently Transports Lipopolysaccharide

Abstract: The presence of an external LPS layer in the outer membrane makes Gram-negative bacteria intrinsically resistant to many antibiotics. Millions of LPS molecules are transported to the cell surface per generation by the Lpt molecular machine made, in E. coli , by seven essential proteins.

Cited by 6 publications

References 41 publications

Dynamic basis of lipopolysaccharide export by LptB₂FGC

Dynamic basis of lipopolysaccharide export by LptB₂FGC

ABC Transporters in Bacterial Nanomachineries

Conformational Investigation of the Asymmetric Periplasmic Domains of E. coli LptB2FGC Using SDSL CW EPR Spectroscopy

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Suppressor Mutations in LptF Bypass Essentiality of LptC by Forming a Six-Protein Transenvelope Bridge That Efficiently Transports Lipopolysaccharide

Abstract: The presence of an external LPS layer in the outer membrane makes Gram-negative bacteria intrinsically resistant to many antibiotics. Millions of LPS molecules are transported to the cell surface per generation by the Lpt molecular machine made, in E. coli , by seven essential proteins.

Cited by 6 publications

References 41 publications

Dynamic basis of lipopolysaccharide export by LptB2FGC

Dynamic basis of lipopolysaccharide export by LptB2FGC

ABC Transporters in Bacterial Nanomachineries

Conformational Investigation of the Asymmetric Periplasmic Domains of E. coli LptB2FGC Using SDSL CW EPR Spectroscopy

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Product

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Dynamic basis of lipopolysaccharide export by LptB₂FGC

Dynamic basis of lipopolysaccharide export by LptB₂FGC