Novel Structure of the Conserved Gram-Negative Lipopolysaccharide Transport Protein A and Mutagenesis Analysis

Suits, M.D.L.; Sperandeo, Paola; Dehò, Gianni; Polissi, Alessandra; Jia, Zongchao

doi:10.1016/j.jmb.2008.04.045

Cited by 146 publications

(230 citation statements)

References 44 publications

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“…LptE is anchored to the inner leaflet of the OM via an N-terminal lipid moiety. To understand the role of this lipid anchor in bringing the LptD/E complex together in vivo, we engineered a plasmid in which the coding sequencing of LptE, excluding its signal sequence and the N-terminal lipidated cysteine (a.a. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19], is placed after the sequence encoding the pelB leader peptide. This plasmid constitutively expresses a soluble version of LptE in the periplasm that is no longer lipidated at the N terminus.…”

Section: Resultsmentioning

confidence: 99%

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Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane

Chng

Ruiz

Chimalakonda

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

185

247

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Lipopolysaccharide (LPS) is the major glycolipid that is present in the outer membranes (OMs) of most Gram-negative bacteria. LPS molecules are assembled with divalent metal cations in the outer leaflet of the OM to form an impervious layer that prevents toxic compounds from entering the cell. For most Gram-negative bacteria, LPS is essential for growth. In Escherichia coli, eight essential proteins have been identified to function in the proper assembly of LPS following its biosynthesis. This assembly process involves release of LPS from the inner membrane (IM), transport across the periplasm, and insertion into the outer leaflet of the OM. Here, we describe the biochemical characterization of the two-protein complex consisting of LptD and LptE that is responsible for the assembly of LPS at the cell surface. We can overexpress and purify LptD and LptE as a stable complex in a 1∶1 stoichiometry. LptD contains a soluble N-terminal domain and a C-terminal transmembrane domain. LptE stabilizes LptD by interacting strongly with the C-terminal domain of LptD. We also demonstrate that LptE binds LPS specifically and may serve as a substrate recognition site at the OM. T he OM of Gram-negative bacteria is a unique asymmetric lipid bilayer in which the outer leaflet is composed almost entirely of LPS and the inner leaflet of phospholipids (PL) (Fig. 1) (1, 2). Building and maintaining this asymmetric bilayer is a challenge for the cell because the OM is located outside the cytoplasm, in an environment that lacks an obvious energy source such as ATP. LPS and PL are synthesized at the cytoplasmic face of the IM whereas lipoproteins and integral membrane proteins are synthesized in the cytoplasm; all these OM components must be transported across the IM and the aqueous periplasmic compartment to be assembled into the OM (3, 4). In the case of LPS assembly, the molecule must ultimately reach the outer leaflet of the OM (Fig.

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Section: Resultsmentioning

confidence: 99%

“…Because LPS is amphipathic, it is unlikely to diffuse across the periplasm unassisted. LptA (formerly YhbN), a periplasmic protein, is thought to mediate LPS transit across this aqueous compartment by an unknown mechanism (9,10,(13)(14)(15). Once LPS arrives at the OM, a protein complex consisting of LptD and LptE (formerly Imp and RlpB, resp.)…”

mentioning

confidence: 99%

Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane

Chng

Ruiz

Chimalakonda

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

185

247

View full text Add to dashboard Cite

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“…The absence of any one of Lpt components prevents LPS transport to the OM [14,15,19]. Only LptA [16] and LptC [18] have been structurally characterized.…”

Section: Introductionmentioning

confidence: 99%

“…doi:10.1016/j.bbrc.2010. 12.121 mature LPS molecule to be released from the IM and transferred to the periplasmic carrier molecule LptA [16]. The small 19.1 kDa protein LptC anchored to the IM through an N-terminal transmembrane helix forms a complex with LptBFG [11].…”

Section: Introductionmentioning

confidence: 99%

“…According to one, a soluble LptA monomer or oligomer chaperones the mature LPS molecule across the periplasm and docks at the LptDE complex in the OM [3,12,16,19].A second mechanism proposes formation of a scaffold consisting of LptA oligomers stacked head-to-tail (fibrils), physically bridging the periplasm with a hydrophobic groove running the entire length of the chain into which LPS could bind [3,12,15,19]. A third mechanism suggests that the LPS transport occurs through membrane contact points, referred to as Bayer junctions [16]. A recent report describing isolation of the trans-envelope complex containing all seven Lpt proteins in a single membrane fraction [3], supports the bridge hypothesis.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of interactions between LPS transport proteins of the Lpt system

Bowyer

Baardsnes

Ajamian

et al. 2011

Biochemical and Biophysical Research Communications

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The lipopolysaccharide transport system (Lpt) in Gram-negative bacteria is responsible for transporting lipopolysaccharide (LPS) from the cytoplasmic surface of the inner membrane, where it is assembled, across the inner membrane, periplasm and outer membrane, to the surface where it is then inserted in the outer leaflet of the asymmetric lipid bilayer. The Lpt system consists of seven known LPS transport proteins (LptA-G) spanning from the cytoplasm to the cell surface. We have shown that the periplasmic component, LptA is able to form a stable complex with the inner membrane anchored LptC but does not interact with the outer membrane anchored LptE. This suggests that the LptC component of the LptBFGC complex may act as a dock for LptA, allowing it to bind LPS after it has been assembled at the inner membrane. That no interaction between LptA and LptE has been observed supports the theory that LptA binds LptD in the LptDE homodimeric complex at the outer membrane.Crown

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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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Novel Structure of the Conserved Gram-Negative Lipopolysaccharide Transport Protein A and Mutagenesis Analysis

Cited by 146 publications

References 44 publications

Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane

Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane

Characterization of interactions between LPS transport proteins of the Lpt system

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

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