Highlights from recently determined structures of membrane proteins: a focus on channels and transporters.

After decades of absent or lackluster growth, recent years have at long last witnessed an exponential growth in the number of novel membrane protein structures determined. Every single achievement has had a tremendous impact on the scientific community, providing an unprecedented wealth of information that typically only an atomic resolution structure can contribute to our molecular understanding of how a protein functions. Presented here is a review of some of the most exciting novel structures of channels and transporters determined by X-ray crystallography in the last two years, and a discussion of their analogies, differences and mechanistic implications

There is a baby in the bath water: AcrB contamination is a major problem in membrane-protein crystallization

Crystals of an E. coli AcrB contaminant were grown from 95% pure CorA preparations. This very frequently occurring problem in membrane-protein crystallography laboratories is reported, as well as suggestions to avoid it

Pollution from livestock farming antibiotics an emerging environmental and human health concern: A review

International audienceVarious studies show that antibiotics have long been used in medicine, particularly in veterinary medicine, as additives in animal food either for the stimulation of their growth or for the therapy of certain infectious diseases. However, others have proven that these antibiotics intensive use and their massive quantity in solid and liquid farming waste have caused many environmental risks. This review paper presents the state of the art, which is specifically related to the impact of the extensive use of some veterinary antibiotics in the breeding of some animals, notably cows, pigs, and chickens. Relevant to the raised issue is the discussion of the pollution of animal manures and agricultural soil by antibiotic residues, which probably result in the multiplication of some antibiotic resistant pathogens. This type of resistance is embodied in the roughly immediate cropping up of infectious diseases in animals, plants, and humans. This review article as an examination of the literature is part of not only fighting against antibiotic resistance but also suggesting some efficient solutions. Among these solutions, physicochemical or biological methods for antibiotics removal were extensively studied. The use of ligninolytic microorganisms and their enzymes were discussed. In this vein, previous studies have found that fungi were active and influential in the antibiotic biodegradation thanks to their enzymes, particularly laccases and peroxidases. Accordingly, this paper sets out to discuss the use of some fungi and their laccases in the biodegradation of some veterinary antibiotics from animal husbandry. This antibiotic biodegradation is a tool to minimize the emergence of antibiotic resistance that is the main purpose in this review

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of C-12 hydroxylase EryK from Saccharopolyspora erythraea

Erythromycin A is produced by Saccharopolyspora erythraea via a secondary metabolic pathway using several steps including glycosylations and hydroxylations of the first macrolide intermediate 6-deoxyerythronolide B. Erythromycin C-12 hydroxylase (CYP113A1), the P450 cytochrome active in the final stages of erythromycin biosynthesis, was cloned and expressed in E. coli. Different crystal forms were harvested from distinct crystallization conditions: two ligand-free forms, one substrate bound and two inhibitors-bound. All crystals belong either to the monoclinc P21 or to the orthorhombic P212121 space groups, and exhibit diffraction limits ranging from 2.3 to 1.6 Å. The structures will be determined by molecular replacement. © 2008 Bentham Science Publishers Ltd

Production and biophysical characterization of the CorA transporter from Methanosarcina mazei.

We report here a general strategy to overproduce and characterize membrane transporters. To illustrate our approach, we selected one member of the CorA transporter family among four tested that belonged to different species. This approach is transposable to other membrane proteins and involves the following steps: (i) cloning by homologous recombination, (ii) high-throughput expression screening, (iii) fermenter-based large-scale production, (iv) high-throughput detergent solubilization screening, (v) protein purification, (vi) multiangle static light scattering/refractometry characterization of purified proteins, (vii) circular dichroism spectroscopy, and (viii) detergent concentration measurements by Fourier transform infrared (FT-IR) spectroscopy. Methanosarcina mazei CorA was expressed in milligram quantities and purified (> 95% pure). n-Dodecyl-beta-D-maltopyranoside (DDM) retained the pentameric native structure of this transporter; thus, we selected it as working detergent. Furthermore, we measured the detergent concentration in our purified and concentrated protein sample by FT-IR to maintain it as low as possible. Our strategy can be adapted to many structural biology approaches as well as for study of single membrane proteins in a variety of conditions

Analysis of the effect of microgravity on protein crystal quality: the case of a myoglobin triple mutant

Crystals of the Met derivative of the sperm whale myoglobin triple mutant Mb-YQR [L(B10)Y, H(E7)Q and T(E10)R] were grown under microgravity conditions and on earth by vapour diffusion. A comparison of crystal quality after complete data collection and processing shows how microgravity-grown crystals diffract to better resolution and lead to considerably improved statistics for X-ray diffraction data compared with crystals grown on earth under the same conditions. The same set of experiments was reproduced on two different Spacelab missions (ISS 6A and ISS 8A) in 2001 and 2002. The structure of this mutant myoglobin, refined using data collected at ELETTRA (Trieste, Italy) from both kinds of crystals, shows that X-ray diffraction from microgravity-grown crystals leads to better defined electron-density maps as well as improved geometrical quality of the refined model. Improvement of the stereochemical parameters of a protein structure is fundamental to quantitative analysis of its function and dynamics and hence to thorough understanding of the molecular mechanisms of action

Functional analysis and crystallographic structure of clotrimazole bound olep, a cytochrome p450 epoxidase from Streptomyces antibioticus involved in oleandomycin biosynthesis

BACKGROUND: OleP is a cyt P450 from Streptomyces antibioticus carrying out epoxigenation of the antibiotic oleandomycin during its biosynthesis. The timing of its reaction has not been fully clarified, doubts remain regarding its substrate and catalytic mechanism. METHODS: The crystal structure of OleP in complex with clotrimazole, an inhibitor of P450s used in therapy, was solved and the complex formation dynamics was characterized by equilibrium and kinetic binding studies and compared to ketoconazole, another azole differing for the N1-substituent. RESULTS: Clotrimazole coordinates the heme and occupies the active site. Most of the residues interacting with clotrimazole are conserved and involved in substrate binding in MycG, the P450 epoxigenase with the highest homology with OleP. Kinetic characterization of inhibitor binding revealed OleP to follow a simple bimolecular reaction, without detectable intermediates. CONCLUSIONS: Clotrimazole-bound OleP adopts an open form, held by a π-π stacking chain that fastens helices F and G and the FG loop. Affinity is affected by the interactions of the N1 substituent within the active site, given the one order of magnitude difference of the off-rate constants between clotrimazole and ketoconazole. Based on structural similarities with MycG, we propose a binding mode for both oleandomycin intermediates, that are the candidate substrates of OleP. GENERAL SIGNIFICANCE: Among P450 epoxigenases OleP is the only one that introduces an epoxide on a non-activated C–C bond. The data here presented are necessary to understand the rare chemistry carried out by OleP, to engineer it and to design more selective and potent P450-targeted drugs

NMR Solution Structure, Backbone Mobility, and Homology Modeling ofc-Type Cytochromes from Gram-Positive Bacteria

International audienceThe solution structure of oxidized cytochrome c553 (71 amino acid residues) from the Gram-positive bacterium Bacillus pasteurii is here reported and compared with the available crystal structure. The solution structure is obtained from 1609 meaningful NOE data (22.7 per residue), 76 dihedral angles, and 59 pseudocontact shifts. The root mean square deviations from the average structure are 0.25±0.07 and 0.59±0.13 Å for the backbone and all heavy atoms, respectively, and the quality assessment of the structure is satisfactory. The solution structure closely reproduces the fold observed in the crystal structure. The backbone mobility was then investigated through amide 15N relaxation rate and 15N–1H NOE measurements. The protein is rigid in both the sub-nanosecond and millisecond time scales, probably due to the relatively large heme:number of amino acids ratio. Modeling of eight c-type cytochromes from other Gram-positive bacteria with a high sequence identity (>30 %) to the present cytochrome c553 was performed. Analysis of consensus features accounts for the relatively low reduction potential as being due to extensive heme hydration and indicates residues 34–35, 44–46, 69–72, and 75 as a conserved hydrophobic patch for the interaction with a protein partner. At variance with mitochondrial c-type cytochrome, this protein does not experience pH-dependent coordination equilibria. The reasons for this difference are analyzed

Crystal Structure of ORF12 from Lactococcus lactis Phage p2 Identifies a Tape Measure Protein Chaperone▿ †

We report here the characterization of the nonstructural protein ORF12 of the virulent lactococcal phage p2, which belongs to the Siphoviridae family. ORF12 was produced as a soluble protein, which forms large oligomers (6- to 15-mers) in solution. Using anti-ORF12 antibodies, we have confirmed that ORF12 is not found in the virion structure but is detected in the second half of the lytic cycle, indicating that it is a late-expressed protein. The structure of ORF12, solved by single anomalous diffraction and refined at 2.9-Å resolution, revealed a previously unknown fold as well as the presence of a hydrophobic patch at its surface. Furthermore, crystal packing of ORF12 formed long spirals in which a hydrophobic, continuous crevice was identified. This crevice exhibited a repeated motif of aromatic residues, which coincided with the same repeated motif usually found in tape measure protein (TMP), predicted to form helices. A model of a complex between ORF12 and a repeated motif of the TMP of phage p2 (ORF14) was generated, in which the TMP helix fitted exquisitely in the crevice and the aromatic patches of ORF12. We suggest, therefore, that ORF12 might act as a chaperone for TMP hydrophobic repeats, maintaining TMP in solution during the tail assembly of the lactococcal siphophage p2

Surfactant Poloxamer 188 as a New Crystallizing Agent for Urate Oxidase

International audienceWe characterize a new class of crystallizing agent for soluble protein crystallization compatible with both pharmaceutical processes and high-resolution structure determination in biocrystallography. Poloxamers are amphiphilic nonionic multiblock polymers used in the cosmetic and pharmaceutical industries. Poloxamer P188 (EO75PO31EO75) is generally used as an emulsifier, solubilizer, and dispersing and wetting agent in the preparation of solid dispersions. Here, we divert the surfactant properties of poloxamer P188 at low concentrations into crystallizing properties at high concentrations of poloxamer P188 to crystallize urate oxidase and to control its crystal design