Probing amino acid side chains of the integral membrane protein PagP by solution NMR: Side chain immobilization facilitates association of secondary structures.

Solution NMR spectroscopy of large protein systems is hampered by rapid signal decay, so most multidimensional studies focus on long-lived H-C magnetization in methyl groups and/or backbone amide H-N magnetization in an otherwise perdeuterated environment. Herein we demonstrate that it is possible to biosynthetically incorporate additional H-C groups that possess long-lived magnetization using cost-effective partially deuterated or unlabeled amino acid precursors added to Escherichia coli growth media. This approach is applied to the outer membrane enzyme PagP in membrane-mimetic dodecylphosphocholine micelles.

Early evolutionary loss of the lipid A modifying enzyme PagP resulting in innate immune evasion in .

Immune evasion through membrane remodeling is a hallmark of pathogenesis. remodels its membrane during its life cycle as it alternates between mammalian hosts (37 °C) and ambient (21 °C to 26 °C) temperatures of the arthropod transmission vector or external environment. This shift in growth temperature induces changes in number and length of acyl groups on the lipid A portion of lipopolysaccharide (LPS) for the enteric pathogens () and (), as well as the causative agent of plague, ().

Polymorphic Regulation of Outer Membrane Lipid A Composition.

The importance of the polymorphic-phase behavior of lipid A structural variations in determining their endotoxic activities has been recognized previously, but any potential role for lipid A polymorphism in controlling outer membrane structure and function has been largely ignored until now. In a recent article in mBio [7(5):e01532-16, https://doi.org/10.

Emerging roles for anionic non-bilayer phospholipids in fortifying the outer membrane permeability barrier.

Lately, researchers have been actively investigating Escherichia coli lptD mutants, which exhibit reduced transport of lipopolysaccharide to the cell surface. In this issue of the Journal of Bacteriology, Sutterlin et al. (H.

Structural biology: Lipopolysaccharide rolls out the barrel.

Two crystal structures of the LptD–LptE protein complex reveal how the cell-wall component lipopolysaccharide is delivered and inserted into the external leaflet of the bacterial outer membrane.

PhoPQ regulates acidic glycerophospholipid content of the Salmonella Typhimurium outer membrane.

Gram-negative bacteria have two lipid membranes separated by a periplasmic space containing peptidoglycan. The surface bilayer, or outer membrane (OM), provides a barrier to toxic molecules, including host cationic antimicrobial peptides (CAMPs). The OM comprises an outer leaflet of lipid A, the bioactive component of lipopolysaccharide (LPS), and an inner leaflet of glycerophospholipids (GPLs).

A divergent Pseudomonas aeruginosa palmitoyltransferase essential for cystic fibrosis-specific lipid A.

Strains of Pseudomonas aeruginosa (PA) isolated from the airways of cystic fibrosis patients constitutively add palmitate to lipid A, the membrane anchor of lipopolysaccharide. The PhoPQ regulated enzyme PagP is responsible for the transfer of palmitate from outer membrane phospholipids to lipid A. This enzyme had previously been identified in many pathogenic Gram-negative bacteria, but in PA had remained elusive, despite abundant evidence that its lipid A contains palmitate.

Detergent-mediated protein aggregation.

Because detergents are commonly used to solvate membrane proteins for structural evaluation, much attention has been devoted to assessing the conformational bias imparted by detergent micelles in comparison to the native environment of the lipid bilayer. Here, we conduct six 500-ns simulations of a system with >600,000 atoms to investigate the spontaneous self assembly of dodecylphosphocholine detergent around multiple molecules of the integral membrane protein PagP. This detergent formed equatorial micelles in which acyl chains surround the protein's hydrophobic belt, confirming existing models of the detergent solvation of membrane proteins.

In vitro evaluation of the potential for resistance development to ceragenin CSA-13.

Objectives: Though most bacteria remain susceptible to endogenous antimicrobial peptides, specific resistance mechanisms are known. As mimics of antimicrobial peptides, ceragenins were expected to retain antibacterial activity against Gram-positive and -negative bacteria, even after prolonged exposure. Serial passaging of bacteria to a lead ceragenin, CSA-13, was performed with representative pathogenic bacteria.

Instability of toxin A subunit of AB(5) toxins in the bacterial periplasm caused by deficiency of their cognate B subunits.

Shiga toxin (STx) belongs to the AB(5) toxin family and is transiently localized in the periplasm before secretion into the extracellular milieu. While producing outer membrane vesicles (OMVs) containing only A subunit of the toxin (STxA), we created specific STx1B- and STx2B-deficient mutants of E. coli O157:H7.

Inscribing the perimeter of the PagP hydrocarbon ruler by site-specific chemical alkylation.

The Escherichia coli outer membrane phospholipid:lipid A palmitoyltransferase PagP selects palmitate chains using its β-barrel-interior hydrocarbon ruler and interrogates phospholipid donors by gating them laterally through an aperture known as the crenel. Lipid A palmitoylation provides antimicrobial peptide resistance and modulates inflammation signaled through the host TLR4/MD2 pathway. Gly88 substitutions can raise the PagP hydrocarbon ruler floor to correspondingly shorten the selected acyl chain.

PagP crystallized from SDS/cosolvent reveals the route for phospholipid access to the hydrocarbon ruler.

Enzymatic reactions involving bilayer lipids occur in an environment with strict physical and topological constraints. The integral membrane enzyme PagP transfers a palmitoyl group from a phospholipid to lipid A in order to assist Escherichia coli in evading host immune defenses during infection. PagP measures the palmitoyl group with an internal hydrocarbon ruler that is formed in the interior of the eight-stranded antiparallel β barrel.

A thiolate anion buried within the hydrocarbon ruler perturbs PagP lipid acyl chain selection.

The Escherichia coli outer membrane phospholipid:lipid A palmitoyltransferase PagP exhibits remarkable selectivity because its binding pocket for lipid acyl chains excludes those differing in length from palmitate by a solitary methylene unit. This narrow detergent-binding hydrophobic pocket buried within the eight-strand antiparallel beta-barrel is known as the hydrocarbon ruler. Gly88 lines the acyl chain binding pocket floor, and its substitution can raise the floor to correspondingly shorten the selected acyl chain.

Molecular mechanism for lateral lipid diffusion between the outer membrane external leaflet and a beta-barrel hydrocarbon ruler.

Membrane-intrinsic enzymes are embedded in lipids, yet how such enzymes interrogate lipid substrates remains a largely unexplored fundamental question. The outer membrane phospholipid:lipid A palmitoyltransferase PagP combats host immune defenses during infection and selects a palmitate chain using its beta-barrel interior hydrocarbon ruler. Both a molecular embrasure and crenel in Escherichia coli PagP display weakened transmembrane beta-strand hydrogen bonding to provide potential lateral routes for diffusion of the palmitoyl group between the hydrocarbon ruler and outer membrane external leaflet.

Structural modifications of outer membrane vesicles to refine them as vaccine delivery vehicles.

In an effort to devise a safer and more effective vaccine delivery system, outer membrane vesicles (OMVs) were engineered to have properties of intrinsically low endotoxicity sufficient for the delivery of foreign antigens. Our strategy involved mutational inactivation of the MsbB (LpxM) lipid A acyltransferase to generate OMVs of reduced endotoxicity from Escherichia coli (E. coli) O157:H7.

Modulation of hexa-acyl pyrophosphate lipid A population under Escherichia coli phosphate (Pho) regulon activation.

Environmental phosphate is an important signal for microorganism gene regulation, and it has recently been shown to trigger some key bacterial virulence mechanisms. In many bacteria, the Pho regulon is the major circuit involved in adaptation to phosphate limitation. The Pho regulon is controlled jointly by the two-component regulatory system PhoR/PhoB and by the phosphate-specific transport (Pst) system, which both belong to the Pho regulon.

PagP activation in the outer membrane triggers R3 core oligosaccharide truncation in the cytoplasm of Escherichia coli O157:H7.

The Escherichia coli outer membrane phospholipid:lipid A palmitoyltransferase PagP is normally a latent enzyme, but it can be directly activated in outer membranes by lipid redistribution associated with a breach in the permeability barrier. We now demonstrate that a lipid A myristate deficiency in an E. coli O157:H7 msbB mutant constitutively activates PagP in outer membranes.

Structural biology of membrane-intrinsic beta-barrel enzymes: sentinels of the bacterial outer membrane.

The outer membranes of Gram-negative bacteria are replete with integral membrane proteins that exhibit antiparallel beta-barrel structures, but very few of these proteins function as enzymes. In Escherichia coli, only three beta-barrel enzymes are known to exist in the outer membrane; these are the phospholipase OMPLA, the protease OmpT, and the phospholipidColon, two colonslipid A palmitoyltransferase PagP, all of which have been characterized at the structural level. Structural details have also emerged for the outer membrane beta-barrel enzyme PagL, a lipid A 3-O-deacylase from Pseudomonas aeruginosa.

Gauging a hydrocarbon ruler by an intrinsic exciton probe.

The structural basis of lipid acyl-chain selection by membrane-intrinsic enzymes is poorly understood because most integral membrane enzymes of lipid metabolism have proven refractory to structure determination; however, robust enzymes from the outer membranes of gram-negative bacteria are now providing a first glimpse at the underlying mechanisms. The methylene unit resolution of the phospholipid:lipid A palmitoyltransferase PagP is determined by the hydrocarbon ruler, a 16-carbon saturated acyl-chain-binding pocket buried within the transmembrane beta-barrel structure. Substitution of Gly88 lining the floor of the hydrocarbon ruler with Ala or Met makes the enzyme select specifically 15- or 12-carbon saturated acyl chains, respectively, indicating that hydrocarbon ruler depth determines acyl-chain selection.

Secondary acylation of Klebsiella pneumoniae lipopolysaccharide contributes to sensitivity to antibacterial peptides.

Klebsiella pneumoniae is an important cause of nosocomial Gram-negative sepsis. Lipopolysaccharide (LPS) is considered to be a major virulence determinant of this encapsulated bacterium and most mutations to the lipid A anchor of LPS are conditionally lethal to the bacterium. We studied the role of LPS acylation in K.