title: Gram-negative trimeric porins have specific LPS binding sites that are essential for porin biogenesis.
abstract: The outer membrane (OM) of gram-negative bacteria is an unusual asymmetric bilayer with an external monolayer of lipopolysaccharide (LPS) and an inner layer of phospholipids. The LPS layer is rigid and stabilized by divalent cation cross-links between phosphate groups on the core oligosaccharide regions. This means that the OM is robust and highly impermeable to toxins and antibiotics. During their biogenesis, OM proteins (OMPs), which function as transporters and receptors, must integrate into this ordered monolayer while preserving its impermeability. Here we reveal the specific interactions between the trimeric porins of Enterobacteriaceae and LPS. Isolated porins form complexes with variable numbers of LPS molecules, which are stabilized by calcium ions. In earlier studies, two high-affinity sites were predicted to contain groups of positively charged side chains. Mutation of these residues led to the loss of LPS binding and, in one site, also prevented trimerization of the porin, explaining the previously observed effect of LPS mutants on porin folding. The high-resolution X-ray crystal structure of a trimeric porin-LPS complex not only helps to explain the mutagenesis results but also reveals more complex, subtle porin-LPS interactions and a bridging calcium ion.
authors: Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom;
authors: Wanatchaporn
authors: W
authors: Arunmanee
authors: Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom;
authors: Monisha
authors: M
authors: Pathania
authors: Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom; Newcastle University Protein and Proteome Analysis, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom;
authors: Alexandra S
authors: AS
authors: Solovyova
authors: National Deuteration Facility, Bragg Institute, Australian Nuclear Science and Technology Organisation, Kirrawee DC, NSW 2232, Australia.
authors: Anton P
authors: AP
authors: Le Brun
authors: Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom;
authors: Helen
authors: H
authors: Ridley
authors: Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom;
authors: Arnaud
authors: A
authors: Baslé
authors: Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom; bert.van-den-berg@newcastle.ac.uk jeremy.lakey@ncl.ac.uk.
authors: Bert
authors: B
authors: van den Berg
authors: Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom; bert.van-den-berg@newcastle.ac.uk jeremy.lakey@ncl.ac.uk.
authors: Jeremy H
authors: JH
authors: Lakey
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002413:Cations, Divalent; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D018272:Porins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D055672:Static Electricity
chemical_list: D002413:Cations, Divalent; D008070:Lipopolysaccharides; C113724:OmpC protein; C109237:OmpF protein; D018272:Porins; D002118:Calcium
keywords: X-ray crystal structure; gram-negative bacteria; lipopolysaccharide; outer membrane; porin