Residues within the LptC transmembrane helix are critical for Escherichia coli LptB2 FG ATPase regulation

Protein Sci. 2024 Feb;33(2):e4879. doi: 10.1002/pro.4879.

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.

Keywords: ABC transporter; EPR spectroscopy; LPS transport; differential scanning fluorimetry; lipopolysaccharide; outer membrane biogenesis.

MeSH terms

  • ATP-Binding Cassette Transporters / metabolism
  • Adenosine Triphosphatases / genetics
  • Adenosine Triphosphatases / metabolism
  • Bacterial Outer Membrane Proteins / metabolism
  • Biological Transport / physiology
  • Carrier Proteins / chemistry
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Escherichia coli Proteins* / chemistry
  • Lipopolysaccharides / metabolism
  • Membrane Proteins / chemistry
  • Periplasmic Proteins* / metabolism

Substances

  • Escherichia coli Proteins
  • Adenosine Triphosphatases
  • Lipopolysaccharides
  • Membrane Proteins
  • Periplasmic Proteins
  • Bacterial Outer Membrane Proteins
  • LptA protein, E coli
  • Carrier Proteins
  • LptB protein, E coli
  • ATP-Binding Cassette Transporters
  • LptC protein, E coli