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.gram-negative bacteria | lipopolysaccharide | X-ray crystal structure | porin | outer membrane

Fosfomycin Permeation through the Outer Membrane Porin OmpF

“…The obtained results will be most interesting for studies of other porins with similar positively charged arginine ladders. Among these porins are the OmpF homologous or semihomologous OM Gram-negative bacterial proteins OmpC (68), PhoE (26), Omp32 (69), OmpK35/36 (70), OmpE35/ 36 (71), OprD (72), OprP (73), and OprO (74). Similar mechanisms as extracted here will likely apply, but especially for the narrower pores like OprD and OprP, the permeation rates will be much smaller than those for OmpF.…”

Localized incorporation of outer membrane components in the pathogen Brucella abortus

“…coli (Rocque et al, 1987;Strittmatter & Galanos, 1987;Buehler et al, 1991;de Cock & Tommassen, 1996), Salmonella (Strittmatter & Galanos, 1987;Latsch et al, 1992;Hagge et al, 2002) or Yersinia (Strittmatter & Galanos, 1987;Vakorina et al, 2003). This is well described for OmpF, a major porin of E. coli (Bolla et al, 1988;Holzenburg et al, 1989;Diedrich et al, 1990;Buehler et al, 1991;Sen & Nikaido, 1991;Arunmanee et al, 2014Arunmanee et al, , 2016Patel et al, 2016) belonging to the same family as Omp2b. One selective advantage of clustering R-LPS and Omp2b could be to facilitate diffusion of compounds from the medium into the pores of the porin, as suggested by theoretical simulations using OmpF as a model (Patel et al, 2016).…”

Polymorphic Regulation of Outer Membrane Lipid A Composition

“…Critical packing parameters are known for most of the glycerophospholipids, but until recently, only the structures of the unmodified hexa-acylated lipid A and its precursor from enterobacteria, lipid IV A , had been cocrystallized in certain membrane protein structures. A recent porin structure from the group of Jeremy Lakey has now revealed a hepta-acylated lipid A partial structure with C 16 esterified to the proximal glucosamine unit (13). As expected, the hepta-acylated lipid A has a larger cross-sectional molecular shape than its hexa-acylated counterpart, and it takes on a perceptible increase in conical shape.…”