Faculty Collaboration Database

Binding and transport of LPS occurs through the coordinated combination of an array of sites across the entire Escherichia coli LPS transport protein LptA. Protein Sci 2023 Aug;32(8):e4724

Date

07/07/2023

Pubmed ID

37417889

Pubmed Central ID

PMC10360375

DOI

10.1002/pro.4724

Scopus ID

2-s2.0-85165512022 (requires institutional sign-in at Scopus site)   1 Citation

Abstract

The outer leaflet of the outer membrane (OM) of bacteria such as Escherichia coli, Pseudomonas aeruginosa, and other important pathogens is largely composed of lipopolysaccharide (LPS), which is essential to nearly all Gram-negative bacteria. LPS is transported to the outer leaflet of the OM through a yet unknown mechanism by seven proteins that comprise the LPS transport system. LptA, the only entirely periplasmic Lpt protein, bridges the periplasmic space between the IM LptB₂ FGC and the OM LptDE complexes. LptA is postulated to protect the hydrophobic acyl chains of LPS as it crosses the hydrophilic periplasm, is essential to cell viability, and contains many conserved residues distributed across the protein. To identify which side chains are required for function of E. coli LptA in vivo, we performed a systematic, unbiased, high-throughput screen of the effect of 172 single alanine substitutions on cell viability utilizing an engineered BL21 derivative with a chromosomal knockout of the lptA gene. Remarkably, LptA is highly tolerant to amino acid substitution with alanine. Only four alanine mutants could not complement the chromosomal knockout; CD spectroscopy showed that these substitutions resulted in proteins with significantly altered secondary structure. In addition, 29 partial loss-of-function mutants were identified that led to OM permeability defects; interestingly, these sites were solely located within β-strands of the central core of the protein and each resulted in misfolding of the protein. Therefore, no single residue within LptA is responsible for LPS binding, supporting previous EPR spectroscopy data indicating that sites across the entire protein work in concert to bind and transport LPS.

Author List

Schultz KM, Schneider JR, Fischer MA, Cina NP, Riegert MO, 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
Bacterial Outer Membrane Proteins
Biological Transport
Carrier Proteins
Escherichia coli
Escherichia coli Proteins
Lipopolysaccharides