3Outer membrane proteins (OMPs) play important roles in Gram-negative bacteria, mitochondria and chloroplasts in nutrition transport, protein import, secretion, and other fundamental biological processes [1][2][3] . Dysfunction of mitochondria outer membrane proteins are linked to disorders such as diabetes, Parkinsons and other neurodegenerative diseases 4,5 . The OMPs are inserted and folded correctly into the outer membrane (OM) by the conserved OMP85 family proteins [6][7][8] , suggesting that similar insertion mechanisms may be used in Gram-negative bacteria, mitochondria and chloroplasts.In Gram-negative bacteria, OMPs are synthesized in the cytoplasm, and are transported across the inner membrane by SecYEG into the periplasm 8,9 . The seventeen kilodalton (kDa) protein (Skp) and the survival factor A (SurA) chaperones escort the unfolded OMPs across the periplasm to the β-barrel assembly machinery (BAM), which is responsible for insertion and assembly of OMPs into the OM 10-12 . InEscherichia coli, the BAM complex consists of BamA and four lipoprotein subunits, BamB, BamC, BamD and BamE. BamA is comprised of five N-terminal polypeptide transport-associated (POTRA) domains and a C-terminal OMP transmembrane barrel, while the four lipoproteins are affixed to the membrane by N-terminal lipid-modified cysteines. Of these subunits, BamA and BamD are essential 3,6 . One copy of each of these five proteins is required to form the BAM complex with an approximate molecular weight of 200 kDa (Extended Data Fig. 1). In vitro reconstitution of the E.coli BAM complex and functional assays showed that all five subunits are required to obtain the maximum activity of BAM [13][14][15][16] . Furthermore, comparison of the two complexes reveals that the periplasmic units are rotated with respect to the barrel, which appears to be linked to significant conformational changes in the β-strands β1C-β6C of the barrel. Taken together this suggests a novel insertion mechanism whereby rotation of the BAM periplasmic ring promotes insertion of OMPs into the OM. To our knowledge, this is the first reported crystal structure of an intramembrane barrel with a lateral-open conformation.Unique architecture of two E. coli BAM complexes X-ray diffraction data of selenomethionine labelled crystals were collected to 3.9Ångström (Å) resolution and the BAM structure was determined by singlewavelength anomalous dispersion (SAD) and manual molecular replacement (Methods, Extended Data Table 1). The first structure contained four proteins: BamA, BamC, BamD and BamE (Fig. 1a-c), with the electron density and crystal packing indicating that the BamB is absent in the complex. This was confirmed by SDS-PAGE analysis of the crystals (Extended Data Fig. 1 and Supplementary Data Fig. S1). In this model, BamA, BamC, BamD and BamE contain residues E22-I806, C25-K344, E26-S243, and C20-E110, respectively. The machinery is approximately 115 Å in length, 84 Å in width and 132 Å in height (Fig. 1a). 5The architecture of BamACDE resembles a top hat with a...

Oligomerisation of membrane proteins in response to lipid binding plays a critical role in many cell-signaling pathways 1 but is often difficult to define 2 or predict 3. Here we develop a mass spectrometry platform to determine simultaneously presence of interfacial lipids and oligomeric stability and discover how lipids act as key regulators of membrane protein association. Evaluation of oligomeric strength for a dataset of 125 α-helical oligomeric membrane proteins revealed an absence of interfacial lipids in the mass spectra of 12 membrane proteins with high oligomeric stability. For the bacterial homologue of the eukaryotic biogenic transporters (LeuT) 4 one of the proteins with the lowest oligomeric stability, we found a precise cohort of lipids within the dimer interface. Delipidation, mutation of lipid binding sites or expression in cardiolipin (CDL) deficient Escherichia coli, abrogated dimer formation. Molecular dynamics simulation revealed that CDL acts as a bidentate ligand bridging across subunits. Subsequently, we show that for the sugar transporter SemiSWEET from Vibrio splendidus 5, another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. We hypothesised that lipids would be essential for dimerisation of the Na + /H + antiporter NhaA from E. coli, which has the lowest oligomeric strength, but not for substantially more stable, homologous NapA from Thermus thermophilus. We found that lipid binding is obligatory for dimerisation of NhaA, whereas NapA has adapted to form an interface that is stable without lipids. Overall, by correlating interfacial strength with the presence of interfacial lipids we provide a rationale for Competing Financial Interests:The authors declare no competing financial interest Data Availability. The raw data for Figure 1 is provided in the Supplementary Table 1. All other data are available upon request. Europe PMC Funders GroupAuthor Manuscript Nature. Author manuscript; available in PMC 2017 July 19. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts understanding the role of lipids in both transient and stable interactions within a range of α-helical membrane proteins, including GPCRs.The recent surge in structure determination of membrane proteins is providing details of protein-lipid binding 6 and yielding insight into the regulatory roles of lipids 7,8. The advent of mass spectrometry (MS) methods for characterising membrane proteins, individually 9, within interactomes 10, and in intact assemblies 11, is adding new information to potential roles of lipids inducing conformational changes 12, contributing to activity and modulating drug efflux (reviewed in 13). The role of lipids towards maintaining the oligomeric state of membrane proteins has however remained widely debated. To understand this phenomenon we performed a bioinformatics analysis of all the α-helical oligomeric transmembrane proteins with known structures. To gauge their relative stability, we ranked these olig...

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.

The twin-arginine translocation (Tat) pathway is one of two general protein transport systems found in the prokaryotic cytoplasmic membrane and is conserved in the thylakoid membrane of plant chloroplasts. The defining, and highly unusual, property of the Tat pathway is that it transports folded proteins, a task that must be achieved without allowing appreciable ion leakage across the membrane. The integral membrane TatC protein is the central component of the Tat pathway. TatC captures substrate proteins by binding their signal peptides. TatC then recruits TatA family proteins to form the active translocation complex. Here we report the crystal structure of TatC from the hyperthermophilic bacterium Aquifex aeolicus. This structure provides a molecular description of the core of the Tat translocation system and a framework for understanding the unique Tat transport mechanism.

SummaryThere has been exponential growth in the number of membrane protein structures determined. Nevertheless, these structures are usually resolved in the absence of their lipid environment. Coarse-grained molecular dynamics (CGMD) simulations enable insertion of membrane proteins into explicit models of lipid bilayers. We have automated the CGMD methodology, enabling membrane protein structures to be identified upon their release into the PDB and embedded into a membrane. The simulations are analyzed for protein-lipid interactions, identifying lipid binding sites, and revealing local bilayer deformations plus molecular access pathways within the membrane. The coarse-grained models of membrane protein/bilayer complexes are transformed to atomistic resolution for further analysis and simulation. Using this automated simulation pipeline, we have analyzed a number of recently determined membrane protein structures to predict their locations within a membrane, their lipid/protein interactions, and the functional implications of an enhanced understanding of the local membrane environment of each protein.