FpvA bound to non-cognate pyoverdines: molecular basis of siderophore recognition by an iron transporter.

The first step in the specific uptake of iron via siderophores in Gram-negative bacteria is the recognition and binding of a ferric siderophore by its cognate receptor. We investigated the molecular basis of this event through structural and biochemical approaches. FpvA, the pyoverdine-Fe transporter from Pseudomonas aeruginosa ATCC 15692 (PAO1 strain), is able to transport ferric-pyoverdines originating from other species, whereas most fluorescent pseudomonads are only able to use the one they produce among the more than 100 known different pyoverdines. We solved the structure of FpvA bound to non-cognate pyoverdines of high- or low-affinity and found a close correlation between receptor-ligand structure and the measured affinities. The structure of the first amino acid residues of the pyoverdine chain distinguished the high- and low-affinity binders while the C-terminal portion of the pyoverdines, often cyclic, does not appear to contribute extensively to the interaction between the siderophore and its transporter. The specificity of the ferric-pyoverdine binding site of FpvA is conferred by the structural elements common to all ferric-pyoverdines, i.e. the chromophore, iron, and its chelating groups.

Mammalian G-protein-coupled receptor expression in Escherichia coli: I. High-throughput large-scale production as inclusion bodies.

G-protein-coupled receptors (GPCRs) represent approximately 3% of human proteome and the most prominent class of pharmacological targets. Despite their important role in many functions, only the X-ray structures of rhodopsin, and more recently of the beta(1)- and beta(2)-adrenergic receptors, have been resolved. Structural studies of GPCRs require that several tedious preliminary steps be fulfilled before setting up the first crystallization experiments: protein expression, detergent solubilization, purification, and stabilization. Here we report on screening expression conditions of approximately 100 GPCRs in Escherichia coli with a view to obtain large amounts of inclusion bodies, a prerequisite to the subsequent refolding step. A set of optimal conditions, including appropriate vectors (Gateway pDEST17oi), strain (C43), and fermentation at high optical density, define the best first instance choice. Beyond this minimal setting, however, the rate of success increases significantly with the number of conditions tested. In contrast with experiments based on a single GPCR expression, our approach provides statistically significant results and indicates that up to 40% of GPCRs can be expressed as inclusion bodies in quantities sufficient for subsequent refolding, solubilization, and purification.

A novel, generic and effective method for the rapid purification of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. Structure determination of G protein-coupled receptors and other applications require milligram quantities of purified receptor proteins on a regular basis. Recombinant GPCRs fused to a heterologous biotinylation domain were produced in the yeast Pichia pastoris. We describe an efficient method for their rapid purification that relies on the capture of these receptors with streptavidin immobilized on agarose beads, and their subsequent release by enzymatic digestion with TEV protease. This method has been applied to several GPCRs belonging to the class A rhodopsin subfamily, leading to high yields of purified proteins; it represents a method of choice for biochemical and biophysical studies when large quantities of purified GPCRs are needed.

A beta strand lock exchange for signal transduction in TonB-dependent transducers on the basis of a common structural motif.

Transport of molecules larger than 600 Da across the outer membrane involves TonB-dependent receptors and TonB-ExbB-ExbD of the inner membrane. The transport is energy consuming, and involves direct interactions between a short N-terminal sequence of receptor, called the TonB box, and TonB. We solved the structure of the ferric pyoverdine (Pvd-Fe) outer membrane receptor FpvA from Pseudomonas aeruginosa in its apo form. Structure analyses show that residues of the TonB box are in a beta strand which interacts through a mixed four-stranded beta sheet with the periplasmic signaling domain involved in interactions with an inner membrane sigma regulator. In this conformation, the TonB box cannot form a four-stranded beta sheet with TonB. The FhuA-TonB or BtuB-TonB structures show that the TonB-FpvA interactions require a conformational change which involves a beta strand lock-exchange mechanism. This mechanism is compatible with movements of the periplasmic domain deduced from crystallographic analyses of FpvA, FpvA-Pvd, and FpvA-Pvd-Fe.

The metal dependence of pyoverdine interactions with its outer membrane receptor FpvA.

To acquire iron, Pseudomonas aeruginosa secretes the fluorescent siderophore pyoverdine (Pvd), which chelates iron and shuttles it into the cells via the specific outer membrane transporter FpvA. We studied the role of iron and other metals in the binding and transport of Pvd by FpvA and conclude that there is no significant affinity between FpvA and metal-free Pvd. We found that the fluorescent in vivo complex of iron-free FpvA-Pvd is in fact a complex with aluminum (FpvA-Pvd-Al) formed from trace aluminum in the growth medium. When Pseudomonas aeruginosa was cultured in a medium that had been treated with a metal affinity resin, the in vivo formation of the FpvA-Pvd complex and the recycling of Pvd on FpvA were nearly abolished. The accumulation of Pvd in the periplasm of Pseudomonas aeruginosa was also reduced in the treated growth medium, while the addition of 1 microM AlCl(3) to the treated medium restored the effects of trace metals observed in standard growth medium. Using fluorescent resonance energy transfer and surface plasmon resonance techniques, the in vitro interactions between Pvd and detergent-solubilized FpvA were also shown to be metal dependent. We demonstrated that FpvA binds Pvd-Fe but not Pvd and that Pvd did not compete with Pvd-Fe for FpvA binding. In light of our finding that the Pvd-Al complex is transported across the outer membrane of Pseudomonas aeruginosa, a model for siderophore recognition based on a metal-induced conformation followed by redox selectivity for iron is discussed.

From the periplasmic signaling domain to the extracellular face of an outer membrane signal transducer of Pseudomonas aeruginosa: crystal structure of the ferric pyoverdine outer membrane receptor.

The pyoverdine outer membrane receptor, FpvA, from Pseudomonas aeruginosa translocates ferric pyoverdine across the outer membrane through an energy consuming mechanism using the proton motive force and the TonB-ExbB-ExbD energy transducing complex from the inner membrane. We solved the crystal structure of the full-length FpvA bound to iron-pyoverdine at 2.7 A resolution. Signal transduction to an anti-sigma protein of the inner membrane and to TonB-ExbB-ExbD involves the periplasmic domain, which displays a beta-alpha-beta fold composed of two alpha-helices sandwiched by two beta-sheets. One iron-pyoverdine conformer is bound at the extracellular face of FpvA, revealing the conformer selectivity of the binding site. The loop that contains the TonB box, involved in interactions with TonB, and connects the signaling domain to the plug domain of FpvA is not defined in the electron density following the binding of ferric pyoverdine. The high flexibility of this loop is probably necessary for signal transduction through the outer membrane.

High-yield expression and purification of soluble forms of the anti-apoptotic Bcl-x(L) and Bcl-2 as TolAIII-fusion proteins.

A method is presented to produce large amounts of Bcl-2 and Bcl-x(L), two anti-apoptotic proteins of considerable biomedical interest. Expression constructs were prepared in which the Escherichia coli protein TolAIII, known to promote over expression of soluble product, was added to the N-terminus of Bcl-2 or Bcl-x(L) proteins, which had their C-terminal hydrophobic anchors deleted. Here the expression of these TolAIII-fusion constructs, followed by a two-step metal-affinity based purification protocol is described. The method delivers at least 20 and 10mg of more than 90% pure TolAIII-Bcl-x(L)DeltaC and TolAIII-Bcl-2(2)DeltaC proteins, respectively, per liter of E. coli cell culture. The proteins are released by proteolysis with thrombin providing > 12 mg of Bcl-x(L)DeltaC or > 6 mg of Bcl-2(2)DeltaC per liter of E. coli cell culture with a purity of more than 95%. Whereas Bcl-x(L)DeltaC is soluble both before and after TolAIII removal, Triton X-100 can significantly increase the extraction of TolAIII- Bcl-2(2)DeltaC from the bacterial cells and its subsequent solubility. Far-UV CD spectroscopy demonstrated that they both have an alpha-helical structure. Fluorescence spectroscopy was used to quantitatively analyze the binding of the respiratory inhibitor antimycin A to recombinant Bcl-2 and Bcl-x(L) proteins as well as the displacement of this ligand from the hydrophobic pocket with BH3 Bad-derived peptide. Purified Bcl-x(L)DeltaC and Bcl-2(2)DeltaC both protect isolated mitochondria from Bax-induced release of cytochrome c. The ensemble of data shows that the expressed proteins are correctly folded and functional. Therefore, the TolAIII-fusion system provides a convenient tool for functional characterization and structural studies of anti-apoptotic proteins.

Enhancing functional production of G protein-coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen.

We have optimized the expression level of 20 mammalian G protein-coupled receptors (GPCRs) in the methylotrophic yeast Pichia pastoris. We found that altering expression parameters, including growth temperature, and supplementation of the culture medium with specific GPCR ligands, histidine, and DMSO increased the amount of functional receptor, as assessed by ligand binding, by more than eightfold over standard expression conditions. Unexpectedly, we found that the overall amount of GPCR proteins expressed, in most cases, varied only marginally between standard and optimized expression conditions. Accordingly, the optimized expression conditions resulted in a marked fractional increase in the ratio of ligand binding-competent receptor to total expressed receptor. The results of this study suggest a general approach for increasing yields of functional mammalian GPCRs severalfold over standard expression conditions by using a set of optimized expression condition parameters that we have characterized for the Pichia expression system. Overall, we have more than doubled the number of GPCR targets that can be produced in our laboratories in sufficient amounts for structural studies.

Crystal structure at high resolution of ferric-pyochelin and its membrane receptor FptA from Pseudomonas aeruginosa.

Pyochelin is a siderophore and virulence factor common to Burkholderia cepacia and several Pseudomonas strains. We describe at 2.0 A resolution the crystal structure of the pyochelin outer membrane receptor FptA bound to the iron-pyochelin isolated from Pseudomonas aeruginosa. One pyochelin molecule bound to iron is found in the protein structure, providing the first three-dimensional structure at the atomic level of this siderophore. The pyochelin molecule provides a tetra-dentate coordination of iron, while the remaining bi-dentate coordination is ensured by another molecule not specifically recognized by the protein. The overall structure of the pyochelin receptor is typical of the TonB-dependent transporter superfamily, which uses the proton motive force from the cytoplasmic membrane through the TonB-ExbB-ExbD energy transducing complex to transport ferric ions across the bacterial outer membrane: a transmembrane 22 beta-stranded barrel occluded by a N-terminal domain that contains a mixed four-stranded beta-sheet. The N-terminal TonB box is disordered in two crystal forms, and loop L8 is found to point towards the iron-pyochelin complex, suggesting that the receptor is in a transport-competent conformation.

The crystal structure of the pyoverdine outer membrane receptor FpvA from Pseudomonas aeruginosa at 3.6 angstroms resolution.

The pyoverdine outer membrane receptor FpvA from Pseudomonas aeruginosa translocates ferric-pyoverdine across the outer membrane via an energy consuming mechanism that involves the inner membrane energy transducing complex of TonB-ExbB-ExbD and the proton motive force. We solved the crystal structure of FpvA loaded with iron-free pyoverdine at 3.6 angstroms resolution. The pyoverdine receptor is folded in two domains: a transmembrane 22-stranded beta-barrel domain occluded by an N-terminal domain containing a mixed four-stranded beta-sheet (the plug). The beta-strands of the barrel are connected by long extracellular loops and short periplasmic turns. The iron-free pyoverdine is bound at the surface of the receptor in a pocket lined with aromatic residues while the extracellular loops do not completely cover the pyoverdine binding site. The TonB box, which is involved in intermolecular contacts with the TonB protein of the inner membrane, is observed in an extended conformation. Comparison of this first reported structure of an iron-siderophore transporter from a bacterium other than Escherichia coli with the known structures of the E.coli TonB-dependent transporters reveals a high structural homology and suggests that a common sensing mechanism exists for the iron-loading status in all bacterial iron siderophore transporters.

Preliminary X-ray investigations of several crystal forms of the ferripyoverdine FpvA outer membrane receptor from Pseudomonas aeruginosa bound to ferripyoverdine.

Ferripyoverdine transport across the outer membrane of Pseudomonas aeruginosa by the pyoverdine receptor FpvA and the transcriptional regulation of FpvA involve interactions of the FpvA N-terminal TonB box and signalling domain with proteins from the inner membrane. Several crystallization conditions of FpvA-Pvd-Fe solubilized in C8E4 detergent were obtained and X-ray data were collected from three crystal forms. The resolution limits range from 3.15 to 2.7 angstroms depending on the crystal form. From preliminary analysis of the electron-density maps, the first full-length structure of an outer membrane receptor including a signalling domain should be determined.

Expression of the human mu opioid receptor in a stable Sf9 cell line.

The cDNA that encodes the human mu opioid receptor (hMOR) has been cloned and expressed in Spodoptera frugiperda (Sf9) cells using a nonlytic vector system. The coding sequence fused to the cleavable glycoprotein signal peptide gp 64, and a C-terminal histidine tag was placed under the transcriptional control of the Orgyia pseudotsugata multicapsid nucleopolyhedrosis virus immediate-early 2 (OpIE2) promoter. Transfected cells were selected using Zeocin resistance and the receptor was constitutively expressed at approximately 12000 receptors per cell. Immunofluorescence images illustrated that more than 75% of the Sf9 cells expressed hMOR at the plasma membrane. This is the first report of the constitutive and heterologous expression of a G protein-coupled receptor in a stably transfected Sf9 cell line, under the control of the OpIE2 promoter.

Using EGFP fusions to monitor the functional expression of GPCRs in the Drosophila Schneider 2 cells.

In combining fluorescence measurements with ligand binding assays, the versatility of the EGFP C-terminally fused to the human mu opioid receptor (EGFP-hMOR) has been exploited to notably improve the expression level of functional G protein-coupled receptors in Drosophila S2 cells. A selected array of efficient optimization approaches is presented herein, ranging from a cell-sorting method, allowing for a substantial enrichment in EGFP-hMOR expressing cells, to the addition of chemical and pharmacological chaperones, significantly enhancing the yield and the activity of the expressed receptors. Consistent with previous studies, significant discrepancies were observed between the total amounts of fluorescent receptors over a limited subpopulation capable of ligand binding, even after expression optimization. Subsequently, membrane isopycnic centrifugation experiments allowed to separate the ligand binding active from the non-active membrane fraction, the latter most probably containing misfolded receptors. Taken together, these results illustrate a coherent set of advantageous productive and preparative methods for the production of GPCRs in the highly valuable Drosophila S2 expression system.

Visualization of interactions between siderophore transporters and the energizing protein TonB by native PAGE.

Horizontal nondenaturing electrophoresis of proteins in polyacrylamide gels was used to observe specific interactions between membrane proteins. The method was particularly well suited for solubilized transporters of the outer membrane of Gram-negative bacteria, and allowed specific complexes of transporter and the inner-membrane protein TonB to be isolated. We have used this method to investigate the interactions between four different outer-membrane transporters, and the TonB proteins from two different organisms. The results show that a stable complex can be isolated on gels for all the proteins studied, but can depend in some cases of the detergent used for solubilization. Furthermore, we observe cross-species interaction as TonB from a given organism can interact with transporters from another organism.

Bioprocess parameters of cell growth and human mu opioid receptor expression in recombinant Drosophila S2 cell cultures in a bioreactor.

The paper describes a recombinant Schneider 2 (rS2) cell culture and protein expression in a bioreactor. S2 cells were transfected with a plasmid containing a fusion protein (human mu opioid receptor, hMOR, and green fluorescent protein, EGFP) under the control of inducible metallothionein promoter. A bioprocess in a bioreactor with 5% dissolved oxygen, 27 degrees C and 120 rpm enabled the cell culture to attain 5.3x10(7 )viable cells/mL at 96 h. The induction decreased the cell multiplication (2.5x10(7) viable cells/mL at 72 h). Glutamine and glucose and low levels of lactate were consumed. A fast recombinant protein synthesis took place and, at 6 h of induction, 2x10(4) receptors/cell could be detected by a functional binding assay. Fluorescence measurements showed a progressive increase of recombinant protein expression with a maximal value of 1.26x10(5) fluo counts/s at 24 h of induction. The data shown in this paper indicate a practical and scaleable cell culture bioprocess procedure for the preparation of recombinant proteins expressed in S2 cells.

Interaction of TonB with the outer membrane receptor FpvA of Pseudomonas aeruginosa.

Pyoverdine-mediated iron uptake by the FpvA receptor in the outer membrane of Pseudomonas aeruginosa is dependent on the inner membrane protein TonB1. This energy transducer couples the proton-electrochemical potential of the inner membrane to the transport event. To shed more light upon this process, a recombinant TonB1 protein lacking the N-terminal inner membrane anchor (TonB(pp)) was constructed. This protein was, after expression in Escherichia coli, purified from the soluble fraction of lysed cells by means of an N-terminal hexahistidine or glutathione S-transferase (GST) tag. Purified GST-TonB(pp) was able to capture detergent-solubilized FpvA, regardless of the presence of pyoverdine or pyoverdine-Fe. Targeting of the TonB1 fragment to the periplasm of P. aeruginosa inhibited the transport of ferric pyoverdine by FpvA in vivo, indicating an interference with endogenous TonB1, presumably caused by competition for binding sites at the transporter or by formation of nonfunctional TonB heterodimers. Surface plasmon resonance experiments demonstrated that the FpvA-TonB(pp) interactions have apparent affinities in the micromolar range. The binding of pyoverdine or ferric pyoverdine to FpvA did not modulate this affinity. Apparently, the presence of either iron or pyoverdine is not essential for the formation of the FpvA-TonB complex in vitro.

Dot-blot immunodetection as a versatile and high-throughput assay to evaluate recombinant GPCRs produced in the yeast Pichia pastoris.

One of the major objectives of the Membrane Protein Network program (MePNet, www.mepnet.org) is to express one hundred G-protein-coupled receptors (GPCRs) in the yeast Pichia pastoris. We have developed an antibody-based assay in order to select for the best behaving clones at each step of the receptor preparation, from expression to solubilization. This assay allowed us to quantify the expression of Flag-tagged GPCRs present in various sample types, from crude P. pastoris extracts to native membrane preparations and detergent solubilised fractions. It combines the specificity of ELISA, the sensitivity of enhanced chemiluminescence detection and the speed of high-throughput screening, and can detect as low as 0.001% (w/w) flag-tagged recombinant receptor present in a crude extract. The method was applied to sort recombinant GPCR clones, to rank receptor expression levels and to screen for detergent solubilization efficiency using the human beta2-adrenergic receptor expressed in P. pastoris as a benchmarking standard.

Semliki Forest virus vectors for overexpression of 101 G protein-coupled receptors in mammalian host cells.

Semliki Forest virus vectors were applied for the evaluation of 101 G protein-coupled receptors in three mammalian cell lines. Western blotting demonstrated that 95 of the 101 tested GPCRs showed positive signals. A large number of the GPCRs were expressed at high levels suggesting receptor yields in the range of 1 mg/L or higher, suitable for structural biology applications. Specific binding assays on a selected number of GPCRs were carried out to compare the correlation between total and functional protein expression. Ligands and additives supplemented to the cell culture medium were evaluated for expression enhancement. Selected GPCRs were also expressed from mutant SFV vectors providing enhanced protein expression and reduced host cell toxicity in attempts to further improve receptor yields.

Structural genomics on membrane proteins: comparison of more than 100 GPCRs in 3 expression systems.

Production of recombinant receptors has been one of the major bottlenecks in structural biology on G protein-coupled receptors (GPCRs). The MePNet (Membrane Protein Network) was established to overexpress a large number of GPCRs in three major expression systems, based on Escherichia coli, Pichia pastoris and Semliki Forest virus (SFV) vectors. Evaluation by immunodetection demonstrated that 50% of a total of 103 GPCRs were expressed in bacterial inclusion bodies, 94% in yeast cell membranes and 95% in SFV-infected mammalian cells. The expression levels varied from low to high and the various GPCR families and subtypes were analyzed for their expressability in each expression system. More than 60% of the GPCRs were expressed at milligram levels or higher in one or several systems, compatible to structural biology applications. Functional activity was determined by binding assays in yeast and mammalian cells and the correlation between immunodetection and binding activity was analyzed.

Crystallization and X-ray diffraction analyses of the outer membrane pyochelin receptor FptA from Pseudomonas aeruginosa.

FptA, the pyochelin outer membrane receptor from Pseudomonas aeruginosa, is a siderophore receptor involved in iron uptake when the bacterium grows under iron limitation. Two crystal forms of the FptA-pyochelin complex were obtained under different crystallization conditions. They belong to space groups P1 and P2(1)2(1)2(1) and data sets were collected for both crystal forms. The triclinic crystals diffract to 3.2 A resolution and the orthorhombic crystals show a 1.9 A resolution limit. A data set at the peak of the iron K edge was also collected at 3.1 A resolution.