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Residues within the LptC transmembrane helix are critical for Escherichia coli LptB2 FG ATPase regulation. Protein Sci 2024 Feb;33(2):e4879

Date

12/22/2023

Pubmed ID

38131105

Pubmed Central ID

PMC10804673

DOI

10.1002/pro.4879

Scopus ID

2-s2.0-85182824310 (requires institutional sign-in at Scopus site)

Abstract

Lipopolysaccharide (LPS) synthesis in Gram-negative bacteria is completed at the outer leaflet of the inner membrane (IM). Following synthesis, seven LPS transport (Lpt) proteins facilitate the movement of LPS to the outer membrane (OM), an essential process that if disrupted at any stage has lethal effects on bacterial viability. LptB2 FG, the IM component of the Lpt bridge system, is a type VI ABC transporter that provides the driving force for LPS extraction from the IM and subsequent transport across a stable protein bridge to the outer leaflet of the OM. LptC is a periplasmic protein anchored to the IM by a single transmembrane (TM) helix intercalating within the lateral gate formed by LptF TM5 and LptG TM1. LptC facilitates the hand-off of LPS from LptB2 FG to the periplasmic protein LptA and has been shown to regulate the ATPase activity of LptB2 FG. Here, using an engineered chromosomal knockout system in Escherichia coli to assess the effects of LptC mutations in vivo, we identified six partial loss of function LptC mutations in the first unbiased alanine screen of this essential protein. To investigate the functional effects of these mutations, nanoDSF (differential scanning fluorimetry) and site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy in combination with an in vitro ATPase assay show that specific residues in the TM helix of LptC destabilize the LptB2 FGC complex and regulate the ATPase activity of LptB.

Author List

Cina NP, Frank DW, Klug CS

Author

Candice S. Klug PhD Professor in the Biophysics department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

ATP-Binding Cassette Transporters
Adenosine Triphosphatases
Bacterial Outer Membrane Proteins
Biological Transport
Carrier Proteins
Escherichia coli
Escherichia coli Proteins
Lipopolysaccharides
Membrane Proteins
Periplasmic Proteins