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The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour-diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 Å resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third-generation synchrotron beamlines.

Obtaining well-ordered crystals is a major hurdle to X-ray structure determination of membrane proteins. To facilitate crystal optimization, we investigated the detergent stability of 24 eukaryotic and prokaryotic membrane proteins, predominantly transporters, using a fluorescent-based unfolding assay. We have benchmarked the stability required for crystallization in small micelle detergents, as they are statistically more likely to lead to high-resolution structures. Using this information, we have been able to obtain well-diffracting crystals for a number of sodium and proton-dependent transporters. By including in the analysis seven membrane proteins for which structures are already known, AmtB, GlpG, Mhp1, GlpT, EmrD, NhaA, and LacY, it was further possible to demonstrate an overall trend between protein stability and structural resolution. We suggest that by monitoring membrane protein stability with reference to the benchmarks described here, greater efforts can be placed on constructs and conditions more likely to yield high-resolution structures., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Membrane protein structural biology is still a largely unconquered area, given that approximately 25% of all proteins are membrane proteins and yet less than 150 unique structures are available. Membrane proteins have proven to be difficult to study owing to their partially hydrophobic surfaces, flexibility and lack of stability. The field is now taking advantage of the high-throughput revolution in structural biology and methods are emerging for effective expression, solubilisation, purification and crystallisation of membrane proteins. These technical advances will lead to a rapid increase in the rate at which membrane protein structures are solved in the near future.

Capsular polysaccharides (CPSs) are essential virulence determinants of many pathogenic bacteria. Escherichia coli group 1 CPSs provide paradigms for widespread surface polysaccharide assembly systems in Gram-negative bacteria. In these systems, complex carbohydrate polymers must be exported across the periplasm and outer membrane to the cell surface. Group 1 CPS export requires oligomers of the outer membrane protein, Wza, for translocation across the outer membrane. Assembly also depends on Wzc, an inner membrane tyrosine autokinase known to regulate export and synthesis of group 1 CPS. Here, we provide a structural view of a complex comprising Wzc and Wza that spans the periplasm, connecting the inner and outer membranes. Examination of transmembrane sections of the complex suggests that the periplasm is compressed at the site of complex formation. An important feature of CPS production is the coupling of steps involved in biosynthesis and export. We propose that the Wza-Wzc complex provides the structural and regulatory core of a larger macromolecular machine. We suggest a mechanism by which CPS may move from the periplasm through the outer membrane.

The K antigenic capsular polysaccharide forms a structural layer, the capsule, on the surfaces of Escherichia coli cells. The capsule provides an important protective covering that helps protect encapsulated bacteria from host immune defenses. The assembly and translocation of the capsule requires proteins in the inner and outer membranes. The inner membrane protein Wzc is a tyrosine autokinase that plays an essential role in what is believed to be a coordinated biosynthesis and secretion process. Mutants lacking Wzc can form K antigen oligosaccharides but are unable to polymerize high molecular weight capsular polymers. Wzc homologs have been identified in exopolymer biosynthesis systems in many different Gram-negative and -positive bacteria. Using single particle averaging on cryo-negatively stained samples, we have produced the first three-dimensional structure of this type of membrane protein in its phosphorylated state at approximately 14 A resolution. Perfluoro-octanoate-PAGE analysis of detergent-solubilized oligomeric Wzc and symmetry analysis of the transmission electron microscopy data clearly demonstrated that Wzc forms a tetrameric complex with C4 rotational symmetry. Viewed from the top of the complex, the oligomer is square with a diameter of approximately 100 A and can be divided into four separate densities. From the side, Wzc is approximately 110 A high and has a distinctive appearance similar to an extracted molar tooth. The upper "crown" region is approximately 55 A high and forms a continuous ring of density. Four unconnected "roots" ( approximately 65 A high) emerge from the underside of the crown. We propose that the crown is formed by protein-protein contacts from the four Wzc periplasmic domains, while each root represents an individual cytoplasmic tyrosine autokinase domain.

Processing of single‐crystal X‐ray diffraction data from area detectors can be separated into two steps. First, raw intensities are obtained by integration of the diffraction images, and then data correction and reduction are performed to determine structure‐factor amplitudes and their uncertainties. The second step considers the diffraction geometry, sample illumination, decay, absorption and other effects. While absorption is only a minor effect in standard macromolecular crystallography (MX), it can become the largest source of uncertainty for experiments performed at long wavelengths. Current software packages for MX typically employ empirical models to correct for the effects of absorption, with the corrections determined through the procedure of minimizing the differences in intensities between symmetry‐equivalent reflections; these models are well suited to capturing smoothly varying experimental effects. However, for very long wavelengths, empirical methods become an unreliable approach to model strong absorption effects with high fidelity. This problem is particularly acute when data multiplicity is low. This paper presents an analytical absorption correction strategy (implemented in new software AnACor) based on a volumetric model of the sample derived from X‐ray tomography. Individual path lengths through the different sample materials for all reflections are determined by a ray‐tracing method. Several approaches for absorption corrections (spherical harmonics correction, analytical absorption correction and a combination of the two) are compared for two samples, the membrane protein OmpK36 GD, measured at a wavelength of λ = 3.54 Å, and chlorite dismutase, measured at λ = 4.13 Å. Data set statistics, the peak heights in the anomalous difference Fourier maps and the success of experimental phasing are used to compare the results from the different absorption correction approaches. The strategies using the new analytical absorption correction are shown to be superior to the standard spherical harmonics corrections. While the improvements are modest in the 3.54 Å data, the analytical absorption correction outperforms spherical harmonics in the longer‐wavelength data (λ = 4.13 Å), which is also reflected in the reduced amount of data being required for successful experimental phasing. [ABSTRACT FROM AUTHOR]

Conjugation is the process by which plasmids, including those that carry antibiotic‐resistance genes, are mobilized from one bacterium (the donor) to another (the recipient). The conjugation efficiency of IncF‐like plasmids relies on the formation of mating‐pair stabilization via intimate interactions between outer membrane proteins on the donor (a plasmid‐encoded TraN isoform) and recipient bacteria. Conjugation of the R100‐1 plasmid into Escherichia coli and Klebsiella pneumoniae (KP) recipients relies on pairing between the plasmid‐encoded TraNα in the donor and OmpW in the recipient. Here, the crystal structure of K. pneumoniae OmpW (OmpWKP) is reported at 3.2 Å resolution. OmpWKP forms an eight‐stranded β‐barrel flanked by extracellular loops. The structures of E. coli OmpW (OmpWEC) and OmpWKP show high conservation despite sequence variability in the extracellular loops. [ABSTRACT FROM AUTHOR]

Members of the ATP‐binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP‐binding cassette in the nucleotide‐binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs. [ABSTRACT FROM AUTHOR]

ATP-binding cassette (ABC) transporters are essential proteins that are found across all kingdoms of life. ABC transporters harness the energy of ATP hydrolysis to drive the import of nutrients inside bacterial cells or the export of toxic compounds or essential lipids across bacteria and eukaryotic membranes. Typically, ABC transporters consist of transmembrane domains (TMDs) and nucleotide-binding domains (NBDs) to bind their substrate and ATP, respectively. The TMDs dictate what ligands can be recognised, whereas the NBDs are the power engine of the ABC transporter, carrying out ATP binding and hydrolysis. It has been proposed that they utilise the alternating access mechanism, inward- to outward-facing conformation, to transport their substrates. Here, we will review the recent progress on the structure determination of eukaryotic and bacterial ABC transporters as well as the novel mechanisms that have also been proposed, that fall out of the alternating access mechanism model. [ABSTRACT FROM AUTHOR]

The ATP-binding cassette (ABC) transporters are primary transporters that couple the energy stored in adenosine triphosphate (ATP) to the movement of molecules across the membrane. ABC transporters can be divided into exporters and importers; importers mediate the uptake of essential nutrients into cells and are found predominantly in prokaryotes whereas exporters transport molecules out of cells or into organelles and are found in all organisms. ABC exporters have been linked with multi-drug resistance in both bacterial and eukaryotic cells. ABC transporters are powered by the hydrolysis of ATP and transport their substrate via the alternating access mechanism, whereby the protein alternates between a conformation in which the substrate-binding site is accessible from the outside of the membrane, outward-facing and one in which it is inward-facing. In this mini-review, the structures of different ABC transporter types in different conformations are presented within the context of the alternating access mechanism and how they have shaped our current understanding of the mechanism of ABC transporters. [ABSTRACT FROM AUTHOR]

Optimising membrane protein production yields in Escherichia coli can be time- and resource-consuming. Here, we present a simple and effective Mem brane protein S ingle sho t a mplification r ecipe: MemStar. This one-shot amplification recipe is based on the E. coli strain Lemo21(DE3), the PASM-5052 auto-induction medium and, contradictorily, an IPTG induction step. Using MemStar, production yields for most bacterial membrane proteins tested were improved to reach an average of 5 mg L −1 per OD 600 unit, which is significantly higher than yields obtained with other common production strategies. With MemStar, we have been able to obtain new structural information for several transporters, including the sodium/proton antiporter NapA. [ABSTRACT FROM AUTHOR]

Abstract: Here, we describe a simple and efficient method for the purification of Escherichia coli outer membrane proteins. We have tested this protocol for the purification of Wza and Osmoporin C (OmpC) proteins. Both proteins were purified to homogeneity, in two steps, by anion exchange and size exclusion chromatography with a final yield of 92.5mg for the Wza protein and 291.5mg for the OmpC protein. The purity of the samples was judged by electrophoretic analysis, mass spectrometry, single particle analysis, three-dimensional (3D) crystallisation and X-ray diffraction. [Copyright &y& Elsevier]

Wza is a highly conserved multimeric outer membrane protein complex required for the surface expression of the serotype K30 group 1 capsular polysaccharide in Escherichia coli. Here we present the first three-dimensional structure of this type of polysaccharide exporter at a 15.5-Å resolution obtained using single particle averaging on a dataset of cryo-negatively stained protein. Previous structural studies on purified Wza have revealed a homo-oligomeric ring structure that is most probably composed of eight subunits. Symmetry analysis of the three-dimensional structure combined with biochemical two- and three-dimensional crystallographic data strongly suggest that Wza is an octameric complex with a C4 quasi-rotational symmetry and is organized as a tetramer of dimeric subunits. Wza is best described as a stack of two 4-Å high rings with differing diameters providing a mushroom-like aspect from the side. The larger ring has a distinctive square shape with a diameter of 115 Å, whereas the smaller is almost circular with a diameter of 90 Å. In the center of the complex and enclosed by the four symmetrical arms is a small elliptical cagelike cavity of ∼40 Å in diameter. The central cavity is effectively sealed at the top and bottom of the complex but has small inter-arm holes when viewed from the side. We discuss the structure of this complex and implications in the surface translocation of cell-surface polysaccharide. [ABSTRACT FROM AUTHOR]

The lasso peptide microcin J25 is known to hijack the siderophore receptor FhuA for initiating internalization. Here, we provide what is to our knowledge the first structural evidence on the recognition mechanism, and our biochemical data show that another closely related lasso peptide cannot interact with FhuA. Our work provides an explanation on the narrow activity spectrum of lasso peptides and opens the path to the development of new antibacterials. [ABSTRACT FROM AUTHOR]

A novel integral membrane lipoprotein, Wza, from Escherichia coli serotype O9a:K30 has been purified and crystallized. Wza is required for the surface expression of the serotype K30 group 1 capsular polysaccharide of E. coli; closely related homologues are found in other bacteria that produce extracellular polysaccharides. The Wza crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 94.6, b = 215.5, c = 218.5 Å. A data set to 3.0 Å with 99.8% completeness and an Rmerge of 10.5% has been collected from a single crystal. [ABSTRACT FROM AUTHOR]

The article presents Molecular mechanism of SbmA, a promiscuous transporter exploited by antimicrobial peptides. Topics discussed include the structures show an outward-open conformation with a large cavity that can accommodate diverse substrates; and propose a molecular mechanism for antibacterial peptide uptake paving the way for creation of narrow-targeted therapeutics.

The lipid bilayer is a dynamic environment that consists of a mixture of lipids with different properties that regulate the function of membrane proteins; these lipids are either annular, masking the protein hydrophobic surface, or specific lipids, essential for protein function. In this study, using tandem mass spectrometry, we have identified specific lipids associated with the Escherichia coli ABC transporter McjD, which translocates the antibacterial peptide MccJ25. Using non-denaturing mass spectrometry, we show that McjD in complex with MccJ25 survives the gas phase. Partial delipidation of McjD resulted in reduced ATPase activity and thermostability as shown by circular dichroism, both of which could be restored upon addition of defined E. coli lipids. We have resolved a phosphatidylglycerol lipid associated with McjD at 3.4 Å resolution, whereas molecular dynamic simulations carried out in different lipid environments assessed the binding of specific lipids to McjD. Combined, our data show a synergistic effect of zwitterionic and negatively charged lipids on the activity of McjD; the zwitterionic lipids provide structural stability to McjD, whereas the negatively charged lipids are essential for its function. [ABSTRACT FROM AUTHOR]