Lipids mediate supramolecular outer membrane protein assembly in bacteria

Abstract

β Barrel outer membrane proteins (OMPs) cluster into supramolecular assemblies that give function to the outer membrane (OM) of Gram-negative bacteria. How such assemblies form is unknown. Here, through photoactivatable cross-linking into the Escherichia coli OM, coupled with simulations, and biochemical and biophysical analysis, we uncover the basis for OMP clustering in vivo. OMPs are typically surrounded by an annular shell of asymmetric lipids that mediate higher-order complexes with neighboring OMPs. OMP assemblies center on the abundant porins OmpF and OmpC, against which low-abundance monomeric β barrels, such as TonB-dependent transporters, are packed. Our study reveals OMP-lipid-OMP complexes to be the basic unit of supramolecular OMP assembly that, by extending across the entire cell surface, couples the requisite multifunctionality of the OM to its stability and impermeability.

A Dual Detergent Strategy to Capture a Bacterial Outer Membrane Proteome in Peptidiscs for Characterization by Mass Spectrometry and Binding Assays

“…Our method may also be useful for studying the specific association of outer membrane lipid species on OMP structure and function. Since the lipid components of the outer membrane play a critical role in mediating antibiotic resistance, this is a promising area for future research. , …”

“…The outer membrane of Gram-negative bacteria forms a formidable barrier that is the cell’s first line of defense against many antimicrobials, including antibiotics. This natural impermeability is conferred by a relatively rigid and asymmetric lipid bilayer, which contains phospholipids on the inner leaflet and glycolipidsknown as lipopolysaccharides (LPS)on the outer leaflet. − In addition to these lipid species, the outer membrane contains two major classes of proteins: (i) β-barrel proteins, which are integral to the lipid bilayer, and (ii) lipoproteins, which are covalently tethered to the membrane via a lipid moiety. ,,− In this study, these two protein classes are termed outer membrane proteins (OMPs) and collectively referred to as the “outer membrane proteome”. The importance of this outer membrane proteome and associated lipid components in restricting cell permeability is underscored by the observation that mutations affecting outer membrane integrity result in decreased bacterial virulence and increased susceptibility to antibiotics. − …”

Mass spectrometry of intact membrane proteins: shifting towards a more native-like context

“…PE and PI lipids were identified by native MS and lipidomics and remodelled into the EM structure with high confidence and their roles were ascribed to stabilising the dimer interface. More recently, native MS combined with chemical crosslinking was used to probe the organisation of bacterial outer membrane proteins and lipids [ 65 ]. The pattern of lipid crosslinks captured by native MS amongst various OmpF mutants reflects that the outermost leaflet of the outer membrane is enriched with lipopolysaccharides while the inner leaflets are mainly composed of phospholipids.…”

Mechanism of outer membrane destabilization by global reduction of protein content

“…Populating the OM outer leaflet with PLs does not restore cell shape or viability under large osmotic shocks One of the essential functions of the bacterial OM is to provide mechanical integrity for withstanding forces from inside and outside of the cell (3,23). The stiffness of the OM is largely determined by molecular interactions among outer membrane components including proteins and LPS molecules and between the OM and the cell wall (2)(3)(4). Based on the altered OM composition of the ΔbamD strain and its suppressors (Fig.…”

“…The OM, an asymmetric bilayer with lipopolysaccharide (LPS) in the outer leaflet and glycerophospholipids (PLs) in the inner leaflet, functions as a selective permeability barrier, allowing passage of small hydrophilic compounds while excluding large and hydrophobic molecules (1). An ordered network of outer membrane proteins (OMPs) with patches of LPS stabilized by electrostatic LPS-LPS and LPS-OMP interactions contribute to the mechanical strength of the cell (2)(3)(4). Perturbation to this essential barrier can result in cell death.…”