Dataset Information

Lipopolysaccharide binding to the periplasmic protein LptA.

ABSTRACT: Lipopolysaccharide (LPS) and the periplasmic protein, LptA, are two essential components of Gram-negative bacteria. LPS, also known as endotoxin, is found asymmetrically distributed in the outer leaflet of the outer membrane of Gram-negative bacteria such as Escherichia coli and plays a role in the organism's natural defense in adverse environmental conditions. LptA is a member of the lipopolysaccharide transport protein (Lpt) family, which also includes LptC, LptDE, and LptBFG2 , that functions to transport LPS through the periplasm to the outer leaflet of the outer membrane after MsbA flips LPS across the inner membrane. It is hypothesized that LPS binds to LptA to cross the periplasm and that the acyl chains of LPS bind to the central pocket of LptA. The studies described here are the first to comprehensively characterize and quantitate the binding of LPS by LptA. Using site-directed spin-labeling electron paramagnetic resonance (EPR) spectroscopy, data were collected for 15 spin-labeled residues in and around the proposed LPS binding pocket on LptA to observe the mobility changes caused by the presence of exogenous LPS and identify the binding location of LPS to LptA. The EPR data obtained suggest a 1:1 ratio for the LPS:LptA complex and allow the first calculation of dissociation constants for the LptA-LPS interaction. The results indicate that the entire protein is affected by LPS binding, the N-terminus unfolds in the presence of LPS, and a mutant LptA protein unable to form oligomers has an altered affinity for LPS.

SUBMITTER: Schultz KM

PROVIDER: S-EPMC5521551 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Lipopolysaccharide binding to the periplasmic protein LptA.

Schultz Kathryn M KM Lundquist Tanner J TJ Klug Candice S CS

Protein science : a publication of the Protein Society 20170430 8

Lipopolysaccharide (LPS) and the periplasmic protein, LptA, are two essential components of Gram-negative bacteria. LPS, also known as endotoxin, is found asymmetrically distributed in the outer leaflet of the outer membrane of Gram-negative bacteria such as Escherichia coli and plays a role in the organism's natural defense in adverse environmental conditions. LptA is a member of the lipopolysaccharide transport protein (Lpt) family, which also includes LptC, LptDE, and LptBFG<sub>2</sub> , tha ...[more]

PMID: 28419595

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Project description:The outer membrane (OM) of Gram-negative bacteria is a highly selective permeability barrier due to its asymmetric structure with lipopolysaccharide (LPS) in the outer leaflet. In Escherichia coli, LPS is transported to the cell surface by the LPS transport (Lpt) system composed of seven essential proteins forming a transenvelope bridge. Transport is powered by the ABC transporter LptB2FGC, which extracts LPS from the inner membrane (IM) and transfers it, through LptC protein, to the periplasmic protein LptA. Then, LptA delivers LPS to the OM LptDE translocon for final assembly at the cell surface. The Lpt protein machinery operates as a single device, since depletion of any component leads to the accumulation of a modified LPS decorated with repeating units of colanic acid at the IM outer leaflet. Moreover, correct machine assembly is essential for LPS transit and disruption of the Lpt complex results in LptA degradation. Due to its vital role in cell physiology, the Lpt system represents a good target for antimicrobial drugs. Thanatin is a naturally occurring antimicrobial peptide reported to cause defects in membrane assembly and demonstrated in vitro to bind to the N-terminal ?-strand of LptA. Since this region is involved in both LptA dimerization and interaction with LptC, we wanted to elucidate the mechanism of inhibition of thanatin and discriminate whether its antibacterial effect is exerted by the disruption of the interaction of LptA with itself or with LptC. For this purpose, we here implemented the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system to probe in vivo the Lpt interactome in the periplasm. With this system, we found that thanatin targets both LptC-LptA and LptA-LptA interactions, with a greater inhibitory effect on the former. We confirmed in vitro the disruption of LptC-LptA interaction using two different biophysical techniques. Finally, we observed that in cells treated with thanatin, LptA undergoes degradation and LPS decorated with colanic acid accumulates. These data further support inhibition or disruption of Lpt complex assembly as the main killing mechanism of thanatin against Gram-negative bacteria.

Dataset Information

Lipopolysaccharide binding to the periplasmic protein LptA.

Publications

Lipopolysaccharide binding to the periplasmic protein LptA.

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