The Escherichia coli Lpt Transenvelope Protein Complex for Lipopolysaccharide Export Is Assembled via Conserved Structurally Homologous Domains

Villa, Riccardo; Martorana, Alessandra M.; Okuda, Suguru; Gourlay, L.J.; Nardini, M.; Sperandeo, Paola; Dehò, Gianni; Bolognesi, Martino; Kahne, Daniel; Polissi, Alessandra

doi:10.1128/jb.02057-12

Cited by 95 publications

(143 citation statements)

References 41 publications

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“…LptC does not seem to be a functional component of the IM ABC transporter, as its association with the LptB 2 FG complex does not affect its ATPase activity (8); on the other hand, it appears relevant for proper IM complex assembly (22). Mutational analyses suggest that the interaction of LptC with the IM LptB 2 FG complex is mediated by the N-terminal region of the ␤ jellyroll, whereas its transmembrane domain appears to be dispensable, as a periplasmic soluble version of LptC and a LptC chimera carrying a heterologous transmembrane segment is functional and proficient in Lpt complex assembly (22).…”

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confidence: 99%

“…Such a ␤-jellyroll fold (the Lpt fold) appears to be a key element in driving the assembly of the Lpt machinery. In fact, through these structurally homologous domains, the C terminus of LptC interacts with the N terminus of LptA, and the C terminus of LptA interacts with the N-terminal periplasmic domain of LptD, thus forming a protein bridge that connects the IM and the OM (12,16,20,22).…”

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confidence: 99%

“…Proper interaction of LptC with LptB 2 FG is necessary for LptA recruitment (22). Interaction of the N-terminal domain of LptD with LptA requires the correct maturation of the LptDE complex that in turns depends on nonconsecutive disulfide bond formation in LptD (16); LptE and the chaperone/protease BepA have been implicated in this process (23).…”

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Functional Interaction between the Cytoplasmic ABC Protein LptB and the Inner Membrane LptC Protein, Components of the Lipopolysaccharide Transport Machinery in Escherichia coli

et al. 2016

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The assembly of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) requires the transenvelope Lpt (lipopolysaccharide transport) complex, made in Escherichia coli of seven essential proteins located in the inner membrane (IM) (LptBCFG), periplasm (LptA), and OM (LptDE). At the IM, LptBFG constitute an unusual ATP binding cassette (ABC) transporter, composed by the transmembrane LptFG proteins and the cytoplasmic LptB ATPase, which is thought to extract LPS from the IM and to provide the energy for its export across the periplasm to the cell surface. LptC is a small IM bitopic protein that binds to LptBFG and recruits LptA via its N-and C-terminal regions, and its role in LPS export is not completely understood. Here, we show that the expression level of lptB is a critical factor for suppressing lethality of deletions in the C-terminal region of LptC and the functioning of a hybrid Lpt machinery that carries Pa-LptC, the highly divergent LptC orthologue from Pseudomonas aeruginosa. We found that LptB overexpression stabilizes C-terminally truncated LptC mutant proteins, thereby allowing the formation of a sufficient amount of stable IM complexes to support growth. Moreover, the LptB level seems also critical for the assembly of IM complexes carrying Pa-LptC which is otherwise defective in interactions with the E. coli LptFG components. Overall, our data suggest that LptB and LptC functionally interact and support a model whereby LptB plays a key role in the assembly of the Lpt machinery. IMPORTANCEThe asymmetric outer membrane (OM) of Gram-negative bacteria contains in its outer leaflet an unusual glycolipid, the lipopolysaccharide (LPS). LPS largely contributes to the peculiar permeability barrier properties of the OM that prevent the entry of many antibiotics, thus making Gram-negative pathogens difficult to treat. In Escherichia coli the LPS transporter (the Lpt machine) is made of seven essential proteins (LptABCDEFG) that form a transenvelope complex. Here, we show that increased expression of the membrane-associated ABC protein LptB can suppress defects of LptC, which participates in the formation of the periplasmic bridge. This reveals functional interactions between these two components and supports a role of LptB in the assembly of the Lpt machine.

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Functional Interaction between the Cytoplasmic ABC Protein LptB and the Inner Membrane LptC Protein, Components of the Lipopolysaccharide Transport Machinery in Escherichia coli

et al. 2016

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“…Since LptA, LptC, and N-LptD all belong to the OstA structural superfamily [6,21,22], and that LptA physically interacts with the N-terminal of LptD [13], the N-LptD protein might be the docking site for LptA at the OM. LptC was predicted to have a single transmembrane helix (Trp 7 -Asp 29 ) and a large soluble domain [11,18], and its C-terminal part is involved in the interactions with LptA [13] and the N-terminal region is responsible for binding to LptBFG [8]. An earlier study suggested that Due to the structural similarity between LptC and the periplasmic loop of LptF, it is possible that LptF is the candidate part for interactions in this complex [17].…”

Section: Lpta Interacts With Both Im and Om Lpt Complexmentioning

confidence: 99%

“…The ATP hydrolysis within LptBFG is thought to provide energy for LPS transport and facilitate the release of mature LPS from the IM and enable its transfer to the periplasmic carrier molecule LptA [5][6][7]. The LptC anchors to the IM through an N-terminal transmembrane helix to form a complex with LptBFG and its periplasm domain was found to be the essential function region [8]. However, its function is not well understood within the complex LptBFGC [5,[9][10][11].…”

Section: Introductionmentioning

confidence: 99%

Characterization of lipopolysaccharide transport protein complex

Xiang

Wang

et al. 2013

Open Life Sciences

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Lipopolysaccharide (LPS) is an essential component of the outer membranes (OM) of most Gram-negative bacteria, which plays a crucial role in protection of the bacteria from toxic compounds and harsh conditions. The LPS is biosynthesized at the cytoplasmic side of inner membrane (IM), and then transported across the aqueous periplasmic compartment and assembled correctly at the outer membrane. This process is accomplished by seven LPS transport proteins (LptA-G), but the transport mechanism remains poorly understood. Here, we present findings by pull down assays in which the periplasmic component LptA interacts with both the IM complex LptBFGC and the OM complex LptDE in vitro, but not with complex LptBFG. Using purified Lpt proteins, we have successfully reconstituted the seven transport proteins as a complex in vitro. In addition, the LptC may play an essential role in regulating the conformation of LptBFG to secure the lipopolysaccharide from the inner membrane. Our results contribute to the understanding of lipopolysaccharide transport mechanism and will provide a platform to study the detailed mechanism of the LPS transport in vitro

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Functional Characterization of E. coli LptC: Interaction with LPS and a Synthetic Ligand

et al. 2014

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Lipopolysaccharide (LPS), the main cell-surface molecular constituent of Gram-negative bacteria, is synthesized in the inner membrane (IM) and transported to the outer membrane (OM) by the Lpt (lipopolysaccharide transport) machinery. Neosynthesized LPS is first flipped by MsbA across the IM, then transported to the OM by seven Lpt proteins located in the IM (LptBCFG), in the periplasm (LptA), and in the OM (LptDE). A functional OM is essential to bacterial viability and requires correct placement of LPS in the outer leaflet. Therefore, LPS biogenesis represents an ideal target for the development of novel antibiotics against Gram-negative bacteria. Although the structures of Lpt proteins have been elucidated, little is known about the mechanism of LPS transport, and few data are available on Lpt–LPS binding. We report here the first determination of the thermodynamic and kinetic parameters of the interaction between LptC and a fluorescent lipo-oligosaccharide (fLOS) in vitro. The apparent dissociation constant (Kd) of the fLOS–LptC interaction was evaluated by two independent methods. The first was based on fLOS capture by resin-immobilized LptC; the second used quenching of LptC intrinsic fluorescence by fLOS in solution. The Kd values by the two methods (71.4 and 28.8 μm, respectively) are very similar, and are of the same order of magnitude as that of the affinity of LOS for the upstream transporter, MsbA. Interestingly, both methods showed that fLOS binding to LptC is mostly irreversible, thus reflecting the fact that LPS can be released from LptC only when energy is supplied by ATP or in the presence of a higher-affinity LptA protein. A fluorescent glycolipid was synthesized: this also interacted irreversibly with LptC, but with lower affinity (apparent Kd=221 μM). This compound binds LptC at the LPS binding site and is a prototype for the development of new antibiotics targeting LPS transport in Gram-negative bacteria.

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The Escherichia coli Lpt Transenvelope Protein Complex for Lipopolysaccharide Export Is Assembled via Conserved Structurally Homologous Domains

Cited by 95 publications

References 41 publications

Functional Interaction between the Cytoplasmic ABC Protein LptB and the Inner Membrane LptC Protein, Components of the Lipopolysaccharide Transport Machinery in Escherichia coli

Functional Interaction between the Cytoplasmic ABC Protein LptB and the Inner Membrane LptC Protein, Components of the Lipopolysaccharide Transport Machinery in Escherichia coli

Characterization of lipopolysaccharide transport protein complex

Functional Characterization of E. coli LptC: Interaction with LPS and a Synthetic Ligand

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