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The use of pressure is an advantageous approach to the study of protein structure and dynamics because it can shift the equilibrium populations of protein conformations toward higher energy states that are not of sufficient population to be observable at atmospheric pressure. Recently, the Hubbell group at the University of California, Los Angeles, reintroduced the application of high pressure to the study of proteins by electron paramagnetic resonance (EPR) spectroscopy. This methodology is possible using X-band EPR spectroscopy due to advances in pressure intensifiers, sample cells, and resonators. In addition to the commercial availability of the pressure generation and sample cells by Pressure Biosciences Inc., a five-loop-four-gap resonator required for the initial high pressure EPR spectroscopy experiments by the Hubbell group, and those reported here, was designed by James S. Hyde and built and modified at the National Biomedical EPR Center. With these technological advances, we determined the effect of pressure on the essential periplasmic lipopolysaccharide (LPS) transport protein from Escherichia coli, LptA, and one of its binding partners, LptC. LptA unfolds from the N-terminus to the C-terminus, binding of LPS does not appreciably stabilize the protein under pressure, and monomeric LptA unfolds somewhat more readily than oligomeric LptA upon pressurization to 2 kbar. LptC exhibits a fold and relative lack of stability upon LPS binding similar to LptA, yet adopts an altered, likely monomeric, folded conformation under pressure with only its C-terminus unraveling. The pressure-induced changes likely correlate with functional changes associated with binding and transport of LPS.

After biosynthesis, bacterial lipopolysaccharides (LPS) are transiently anchored to the outer leaflet of the inner membrane (IM). The ATP-binding cassette (ABC) transporter LptB2FG extracts LPS molecules from the IM and transports them to the outer membrane. Here we report the crystal structure of nucleotide-free LptB2FG from Pseudomonas aeruginosa. The structure reveals that lipopolysaccharide transport proteins LptF and LptG each contain a transmembrane domain (TMD), a periplasmic β-jellyroll-like domain and a coupling helix that interacts with LptB on the cytoplasmic side. The LptF and LptG TMDs form a large outward-facing V-shaped cavity in the IM. Mutational analyses suggest that LPS may enter the central cavity laterally, via the interface of the TMD domains of LptF and LptG, and is expelled into the β-jellyroll-like domains upon ATP binding and hydrolysis by LptB. These studies suggest a mechanism for LPS extraction by LptB2FG that is distinct from those of classical ABC transporters that transport substrates across the IM.

A protein complex that moves molecules called lipopolysaccharides between the two cell-wall membranes of certain bacteria is a target for antibiotics. Structures now reveal how this complex delivers its load irreversibly. The molecular processes that move glycolipids between bacterial membranes.

Lipopolysaccharide, an essential component of the Gram-negative bacteria outer membrane, is inserted by LptD–LptE, a protein complex with a unique ‘barrel and plug’ architecture; the structure of the LptD–LptE complex of Shigella flexneri determined here shows LptD forming a 26-stranded β-barrel with LptE located inside the barrel of LptD, the first two β-strands are distorted by two proline residues, creating a potential portal in the barrel wall that might allow lateral diffusion of lipopolysaccharide into the outer membrane. Haohao Dong et al. and Shuai Qiao et al. present X-ray crystal structures of the complex between the lipopolysaccharide transport proteins LptD and LptE from the bacteria Salmonella typhimurium and Shigella flexneri, respectively. The two papers report a unique two-protein plug-and-barrel architecture for the LptD–LptE complex that reveals the mechanism by which the cell-wall lipopolysaccharide is delivered and inserted into the external leaflet of the outer membrane of Gram-negative bacteria. As well as providing new detail on the nature of outer membrane biogenesis, this work will provide data relevant to the design of new antibiotic strategies targeting the bacterial outer membrane, much needed in the fight against multi-drug resistant pathogens. One of the fundamental properties of biological membranes is the asymmetric distribution of membrane lipids. In Gram-negative bacteria, the outer leaflet of the outer membrane is composed predominantly of lipopolysaccharides (LPS)1. The export of LPS requires seven essential lipopolysaccharide transport (Lpt) proteins to move LPS from the inner membrane, through the periplasm to the surface2. Of the seven Lpt proteins, the LptD–LptE complex is responsible for inserting LPS into the external leaflet of the outer membrane3,4. Here we report the crystal structure of the ∼110-kilodalton membrane protein complex LptD–LptE from Shigella flexneri at 2.4 A resolution. The structure reveals an unprecedented two-protein plug-and-barrel architecture with LptE embedded into a 26-stranded β-barrel formed by LptD. Importantly, the secondary structures of the first two β-strands are distorted by two proline residues, weakening their interactions with neighbouring β-strands and creating a potential portal on the barrel wall that could allow lateral diffusion of LPS into the outer membrane. The crystal structure of the LptD–LptE complex opens the door to new antibiotic strategies targeting the bacterial outer membrane.

Lipopolysaccharide (LPS) is essential for most Gram-negative bacteria and has crucial roles in protection of the bacteria from harsh environments and toxic compounds, including antibiotics. Seven LPS transport proteins (that is, LptA–LptG) form a trans-envelope protein complex responsible for the transport of LPS from the inner membrane to the outer membrane, the mechanism for which is poorly understood. Here we report the first crystal structure of the unique integral membrane LPS translocon LptD–LptE complex. LptD forms a novel 26-stranded β-barrel, which is to our knowledge the largest β-barrel reported so far. LptE adopts a roll-like structure located inside the barrel of LptD to form an unprecedented two-protein ‘barrel and plug’ architecture. The structure, molecular dynamics simulations and functional assays suggest that the hydrophilic O-antigen and the core oligosaccharide of the LPS may pass through the barrel and the lipid A of the LPS may be inserted into the outer leaflet of the outer membrane through a lateral opening between strands β1 and β26 of LptD. These findings not only help us to understand important aspects of bacterial outer membrane biogenesis, but also have significant potential for the development of novel drugs against multi-drug resistant pathogenic bacteria.\ud \ud