Structural features of chloroplast trigger factor determined at 2.6†Šresolution.

The folding of newly synthesized polypeptides requires the coordinated action of molecular chaperones. Prokaryotic cells and the chloroplasts of plant cells possess the ribosome-associated chaperone trigger factor, which binds nascent polypeptides at their exit stage from the ribosomal tunnel. The structure of bacterial trigger factor has been well characterized and it has a dragon-shaped conformation, with flexible domains responsible for ribosome binding, peptidyl-prolyl cis-trans isomerization (PPIase) activity and substrate protein binding. Chloroplast trigger-factor sequences have diversified from those of their bacterial orthologs and their molecular mechanism in plant organelles has been little investigated to date. Here, the crystal structure of the plastidic trigger factor from the green alga Chlamydomonas reinhardtii is presented at 2.6 Šresolution. Due to the high intramolecular flexibility of the protein, diffraction to this resolution was only achieved using a protein that lacked the N-terminal ribosome-binding domain. The eukaryotic trigger factor from C. reinhardtii exhibits a comparable dragon-shaped conformation to its bacterial counterpart. However, the C-terminal chaperone domain displays distinct charge distributions, with altered positioning of the helical arms and a specifically altered charge distribution along the surface responsible for substrate binding. While the PPIase domain shows a highly conserved structure compared with other PPIases, its rather weak activity and an unusual orientation towards the C-terminal domain points to specific adaptations of eukaryotic trigger factor for function in chloroplasts.

Structural comparison of the cytochrome P450 enzymes CYP106A1 and CYP106A2 provides insight into their differences in steroid conversion.

Understanding the structural basis of the selectivity of steroid hydroxylation requires detailed structural and functional investigations on various steroid hydroxylases with different selectivities, such as the bacterial cytochrome P450 enzymes. Here, the crystal structure of the cytochrome P450 CYP106A1 from Priestia megaterium was solved. CYP106A1 exhibits a rare additional structural motif of a cytochrome P450, a sixth ß-sheet. The protein was found in different unusual conformations corresponding to both open and closed forms even when crystallized without any known substrate. The structural comparison of CYP106A1 with the previously investigated CYP106A2, including docking studies for both isoforms with the substrate cortisol, reveals a completely different orientation of the steroid molecule in the active sites. This distinction convincingly explains the experimentally observed differences in substrate conversion and product formation by the two enzymes.

Ciliary Proteins Repurposed by the Synaptic Ribbon: Trafficking Myristoylated Proteins at Rod Photoreceptor Synapses.

The Unc119 protein mediates transport of myristoylated proteins to the photoreceptor outer segment, a specialized primary cilium. This transport activity is regulated by the GTPase Arl3 as well as by Arl13b and Rp2 that control Arl3 activation/inactivation. Interestingly, Unc119 is also enriched in photoreceptor synapses and can bind to RIBEYE, the main component of synaptic ribbons. In the present study, we analyzed whether the known regulatory proteins, that control the Unc119-dependent myristoylated protein transport at the primary cilium, are also present at the photoreceptor synaptic ribbon complex by using high-resolution immunofluorescence and immunogold electron microscopy. We found Arl3 and Arl13b to be enriched at the synaptic ribbon whereas Rp2 was predominantly found on vesicles distributed within the entire terminal. These findings indicate that the synaptic ribbon could be involved in the discharge of Unc119-bound lipid-modified proteins. In agreement with this hypothesis, we found Nphp3 (Nephrocystin-3), a myristoylated, Unc119-dependent cargo protein enriched at the basal portion of the ribbon in close vicinity to the active zone. Mutations in Nphp3 are known to be associated with Senior-Løken Syndrome 3 (SLS3). Visual impairment and blindness in SLS3 might thus not only result from ciliary dysfunctions but also from malfunctions of the photoreceptor synapse.

Proton-Coupled Electron Transport in Two Distinct CYBASC Paralogs of Arabidopsis thaliana: A Comparative Characterization of Highly Conserved Tyrosine and Lysine Residues.

CYBASC proteins are ascorbate (AscH-) reducible, diheme b-containing integral membrane cytochrome b561 proteins (cytb561), which are proposed to be involved in AscH- recycling and facilitation of iron absorption. Two distinct CYBASC paralogs from the plant Arabidopsis thaliana, Atcytb561-A (A-paralog) and Atcytb561-B (B-paralog), have been found to differ in their visible-spectral characteristics and their interaction with AscH- and ferric iron chelates. A previously determined crystal structure of the B-paralog provides the first insights into the structural organization of a CYBASC member and implies hydrogen bonding between the substrate AscH- and the conserved lysine residues at positions 77 (B-K77) and 81 (B-K81). The function of the highly conserved tyrosine at position 70 (B-Y70) is not obvious in the crystal structure, but its localization indicates the possible involvement in proton-coupled electron transfer. Here we show that B-Y70 plays a major role in the modulation of the oxidation-reduction midpoint potential of the high-potential heme, EM(bH), as well as in AscH- oxidation. Our results support the involvement of the functionally conserved B-K77 in the stabilization of the dianion Asc2-. These findings are supported by the crystal structure of the B-paralog, but a comparative biochemical and biophysical characterization of the A- and B-paralogs implied distinct and more complex functions of the corresponding residues A-Y69 and A-K76 in the A-paralog. Our results emphasize the need for a high-resolution crystal structure of the A-paralog to illuminate the differences in functional organization between the two paralogs.

Structural and molecular comparison of bacterial and eukaryotic trigger factors.

A considerably small fraction of approximately 60-100 proteins of all chloroplast proteins are encoded by the plastid genome. Many of these proteins are major subunits of complexes with central functions within plastids. In comparison with other subcellular compartments and bacteria, many steps of chloroplast protein biogenesis are not well understood. We report here on the first study of chloroplast-localised trigger factor. In bacteria, this molecular chaperone is known to associate with translating ribosomes to facilitate the folding of newly synthesized proteins. Chloroplast trigger factors of the unicellular green algae Chlamydomonas reinhardtii and the vascular land plant Arabidopsis thaliana were characterized by biophysical and structural methods and compared to the Escherichia coli isoform. We show that chloroplast trigger factor is mainly monomeric and displays only moderate stability against thermal unfolding even under mild heat-stress conditions. The global shape and conformation of these proteins were determined in solution by small-angle X-ray scattering and subsequent ab initio modelling. As observed for bacteria, plastidic trigger factors have a dragon-like structure, albeit with slightly altered domain arrangement and flexibility. This structural conservation despite low amino acid sequence homology illustrates a remarkable evolutionary robustness of chaperone conformations across various kingdoms of life.

Structural characterization of CYP260A1 from Sorangium cellulosum to investigate the 1α-hydroxylation of a mineralocorticoid.

In this study, we report the crystal structure of the cytochrome P450 CYP260A1 (PDB 5LIV) from the myxobacterium Sorangium cellulosum So ce56. In addition, we investigated the hydroxylation of 11-deoxycorticosterone by CYP260A1 by reconstituting the enzyme with the surrogate redox partners adrenodoxin and adrenodoxin reductase. The major product of this steroid conversion was identified as 1α-hydroxy-11-deoxycorticosterone, a novel Δ4 C-21 steroidal derivative. Furthermore, we docked the substrate into the crystal structure and replaced Ser326, the residue responsible for substrate orientation, with asparagine and observed that the mutant S326N displayed higher activity and selectivity for the formation of 1α-hydroxy-11-deoxycorticosterone compared to the wild-type CYP260A1. Thus, our findings highlight the usefulness of the obtained crystal structure of CYP260A1 in identifying biotechnologically more efficient reactions.

Structure-function analysis for the hydroxylation of Δ4 C21-steroids by the myxobacterial CYP260B1.

Myxobacterial CYP260B1 from Sorangium cellulosum was heterologously expressed in Escherichia coli and purified. The in vitro conversion of a small focused substrate library comprised of Δ4 C21-steroids and steroidal drugs using surrogate bovine redox partners shows that CYP260B1 is a novel steroid hydroxylase. CYP260B1 performs the regio- and stereoselective hydroxylation of the glucocorticoid cortodoxone (RSS) to produce 6ß-OH-RSS. The substrate-free crystal structure of CYP260B1 (PDB 5HIW) was resolved. Docking of the tested ligands into the crystal structure suggested that the C17 hydroxy moiety and the presence of either a keto or a hydroxy group at C11 determine the selectivity of hydroxylation.

Crystal Structure of CYP106A2 in Substrate-Free and Substrate-Bound Form.

CYP106A2 from Bacillus megaterium ATCC 13368 is known as a bacterial steroid hydroxylase that is also capable of hydroxylating a variety of terpenoids. To analyze the substrate specificity of this enzyme further, different resin acids of the abietane and pimarane types were tested with regard to binding and conversion. Product formation could be shown for all tested substrates. Spectroscopic studies revealed type I binding spectra for isopimaric acid, but dehydroabietic acid did not induce a high-spin shift of the enzyme. Interestingly, binding of abietic acid resulted in a type II difference spectrum typical for nitrogenous inhibitors. Co-crystallization of CYP106A2 with abietic acid and structure determination revealed bending of the heme cofactor when abietic acid was bound in the active site. Quantum chemical calculations strongly suggest that this heme distortion is the cause of the unusual spectroscopic characteristics.

Reversal of Mitochondrial Transhydrogenase Causes Oxidative Stress in Heart Failure.

Mitochondrial reactive oxygen species (ROS) play a central role in most aging-related diseases. ROS are produced at the respiratory chain that demands NADH for electron transport and are eliminated by enzymes that require NADPH. The nicotinamide nucleotide transhydrogenase (Nnt) is considered a key antioxidative enzyme based on its ability to regenerate NADPH from NADH. Here, we show that pathological metabolic demand reverses the direction of the Nnt, consuming NADPH to support NADH and ATP production, but at the cost of NADPH-linked antioxidative capacity. In heart, reverse-mode Nnt is the dominant source for ROS during pressure overload. Due to a mutation of the Nnt gene, the inbred mouse strain C57BL/6J is protected from oxidative stress, heart failure, and death, making its use in cardiovascular research problematic. Targeting Nnt-mediated ROS with the tetrapeptide SS-31 rescued mortality in pressure overload-induced heart failure and could therefore have therapeutic potential in patients with this syndrome.

An unconventional anaerobic membrane protein production system based on Wolinella succinogenes.

In cases where membrane protein production attempts in more conventional Escherichia coli-based systems have failed, a solution is to resort to a system based on the nonpathogenic epsilon-proteobacterium Wolinella succinogenes. This approach has been demonstrated to be successful for structural and mechanistic analyses not only for homologous production of W. succinogenes membrane proteins but also for the heterologous production of membrane protein complexes from the human pathogens Helicobacter pylori and Campylobacter jejuni. The procedure to establish a system for the production of native and variant enzymes in W. succinogenes is presented in detail for the examples of the quinol:fumarate reductase and the SdhABE complexes of W. succinogenes. Subsequently, further projects using W. succinogenes as expression host are covered.

Replacement of highly conserved E222 by the photostable non-photoconvertible histidine in GFP.

The widely used green fluorescent protein (GFP) decarboxylates upon irradiation; this involves removal of the acidic function of the glutamic acid at position 222, thereby resulting in the irreversible photoconversion of GFP. To suppress this phenomenon, the photostable, non-photoconvertible histidine was introduced at position 222 in GFP. The variant E222H shows negligible photodynamic processes and high expression yield. In addition, the stable and bright fluorescence over a wide pH range makes the E222H protein an alternative for GFP in fluorescence imaging and spectroscopy. Other fluorescent proteins are predicted to benefit from replacement of the catalytic glutamic acid by histidine.

Design, synthesis, and biological testing of novel naphthoquinones as substrate-based inhibitors of the quinol/fumarate reductase from Wolinella succinogenes.

Novel naphthoquinones were designed, synthesized, and tested as substrate-based inhibitors against the membrane-embedded protein quinol/fumarate reductase (QFR) from Wolinella succinogenes, a target closely related to QFRs from the human pathogens Helicobacter pylori and Campylobacter jejuni. For a better understanding of the hitherto structurally unexplored substrate binding pocket, a structure-activity relationship (SAR) study was carried out. Analogues of lawsone (2-hydroxy-1,4-naphthoquinone 3a) were synthesized that vary in length and size of the alkyl side chains (3b-k). A combined study on the prototropic tautomerism of 2-hydroxy-1,4-naphthoquinones series indicated that the 1,4-tautomer is the more stable and biologically relevant isomer and that the presence of the hydroxyl group is crucial for inhibition. Furthermore, 2-bromine-1,4-naphthoquinone (4a-c) and 2-methoxy-1,4-naphthoquinone (5a-b) series were also discovered as novel and potent inhibitors. Compounds 4a and 4b showed IC50 values in low micromolar range in the primary assay and no activity in the counter DT-diaphorase assay.

Crystallization of membrane proteins.

The crystallization of membrane proteins is an essential technique for the determination of atomic models of three-dimensional structures by X-ray crystallography. The compositions of solutions of purified membrane proteins are altered, so as to transiently induce supersaturation, a requirement for crystal nucleation and growth. The establishment of the precise optimal crystallization conditions has to be performed individually by a combination of systematic approaches and trial-and-error. These procedures have become more efficient due to the introduction of laboratory automation. Here we describe the crystallization of the dihaem-containing quinol:fumarate reductase (QFR) membrane protein complex and illustrate key factors important in the screening process.

The di-heme family of respiratory complex II enzymes.

The di-heme family of succinate:quinone oxidoreductases is of particular interest, because its members support electron transfer across the biological membranes in which they are embedded. In the case of the di-heme-containing succinate:menaquinone reductase (SQR) from Gram-positive bacteria and other menaquinone-containing bacteria, this results in an electrogenic reaction. This is physiologically relevant in that it allows the transmembrane electrochemical proton potential Δp to drive the endergonic oxidation of succinate by menaquinone. In the case of the reverse reaction, menaquinol oxidation by fumarate, catalysed by the di-heme-containing quinol:fumarate reductase (QFR), evidence has been obtained that this electrogenic electron transfer reaction is compensated by proton transfer via a both novel and essential transmembrane proton transfer pathway ("E-pathway"). Although the reduction of fumarate by menaquinol is exergonic, it is obviously not exergonic enough to support the generation of a Δp. This compensatory "E-pathway" appears to be required by all di-heme-containing QFR enzymes and results in the overall reaction being electroneutral. In addition to giving a brief overview of progress in the characterization of other members of this diverse family, this contribution summarizes key evidence and progress in identifying constituents of the "E-pathway" within the framework of the crystal structure of the QFR from the anaerobic epsilon-proteobacterium Wolinella succinogenes at 1.78Å resolution. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease.

Hydrogen-bonded networks along and bifurcation of the E-pathway in quinol:fumarate reductase.

The E-pathway of transmembrane proton transfer has been demonstrated previously to be essential for catalysis by the diheme-containing quinol:fumarate reductase (QFR) of Wolinella succinogenes. Two constituents of this pathway, Glu-C180 and heme b(D) ring C (b(D)-C-) propionate, have been validated experimentally. Here, we identify further constituents of the E-pathway by analysis of molecular dynamics simulations. The redox state of heme groups has a crucial effect on the connectivity patterns of mobile internal water molecules that can transiently support proton transfer from the b(D)-C-propionate to Glu-C180. The short H-bonding paths formed in the reduced states can lead to high proton conduction rates and thus provide a plausible explanation for the required opening of the E-pathway in reduced QFR. We found evidence that the b(D)-C-propionate group is the previously postulated branching point connecting proton transfer to the E-pathway from the quinol-oxidation site via interactions with the heme b(D) ligand His-C44. An essential functional role of His-C44 is supported experimentally by site-directed mutagenesis resulting in its replacement with Glu. Although the H44E variant enzyme retains both heme groups, it is unable to catalyze quinol oxidation. All results obtained are relevant to the QFR enzymes from the human pathogens Campylobacter jejuni and Helicobacter pylori.

Heterologous production and characterisation of two distinct dihaem-containing membrane integral cytochrome b(561) enzymes from Arabidopsis thaliana in Pichia pastoris and Escherichia coli cells.

Cytochrome (cyt) b(561) proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b(561)-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b(561) paralogs from Arabidopsis thaliana (Acytb(561)-A, Acytb(561)-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb(561)-A resembles the best characterised member of the CYBASC family, the cytochrome b(561) from adrenomedullary chromaffin vesicles, and that Acytb(561)-B is atypical compared to other CYBASC proteins. Haem oxidation-reduction midpoint potential (E(M)) values were found to be fully consistent with ascorbate oxidation activities and Fe(3+)-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b(561) from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem E(M) values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem E(M) values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe(3+)-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem E(M) values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b(561) paralogs exist as homodimers.

Limited reversibility of transmembrane proton transfer assisting transmembrane electron transfer in a dihaem-containing succinate:quinone oxidoreductase.

Membrane protein complexes can support both the generation and utilisation of a transmembrane electrochemical proton potential (Deltap), either by supporting transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane or by supporting transmembrane proton transfer. The first mechanism has been unequivocally demonstrated to be operational for Deltap-dependent catalysis of succinate oxidation by quinone in the case of the dihaem-containing succinate:menaquinone reductase (SQR) from the Gram-positive bacterium Bacillus licheniformis. This is physiologically relevant in that it allows the transmembrane potential Deltap to drive the endergonic oxidation of succinate by menaquinone by the dihaem-containing SQR of Gram-positive bacteria. In the case of a related but different respiratory membrane protein complex, the dihaem-containing quinol:fumarate reductase (QFR) of the epsilon-proteobacterium Wolinella succinogenes, evidence has been obtained that both mechanisms are combined, so as to facilitate transmembrane electron transfer by proton transfer via a both novel and essential compensatory transmembrane proton transfer pathway ("E-pathway"). Although the reduction of fumarate by menaquinol is exergonic, it is obviously not exergonic enough to support the generation of a Deltap. This compensatory "E-pathway" appears to be required by all dihaem-containing QFR enzymes and results in the overall reaction being electroneutral. However, here we show that the reverse reaction, the oxidation of succinate by quinone, as catalysed by W. succinogenes QFR, is not electroneutral. The implications for transmembrane proton transfer via the E-pathway are discussed.

The correlation of cathodic peak potentials of vitamin K(3) derivatives and their calculated electron affinities. The role of hydrogen bonding and conformational changes.

2-methyl-1,4-naphtoquinone 1 (vitamin K(3), menadione) derivatives with different substituents at the 3-position were synthesized to tune their electrochemical properties. The thermodynamic midpoint potential (E(1/2)) of the naphthoquinone derivatives yielding a semi radical naphthoquinone anion were measured by cyclic voltammetry in the aprotic solvent dimethoxyethane (DME). Using quantum chemical methods, a clear correlation was found between the thermodynamic midpoint potentials and the calculated electron affinities (E(A)). Comparison of calculated and experimental values allowed delineation of additional factors such as the conformational dependence of quinone substituents and hydrogen bonding which can influence the electron affinities (E(A)) of the quinone. This information can be used as a model to gain insight into enzyme-cofactor interactions, particularly for enzyme quinone binding modes and the electrochemical adjustment of the quinone motif.

Production, characterization and determination of the real catalytic properties of the putative 'succinate dehydrogenase' from Wolinella succinogenes.

Both the genomes of the epsilonproteobacteria Wolinella succinogenes and Campylobacter jejuni contain operons (sdhABE) that encode for so far uncharacterized enzyme complexes annotated as 'non-classical' succinate:quinone reductases (SQRs). However, the role of such an enzyme ostensibly involved in aerobic respiration in an anaerobic organism such as W. succinogenes has hitherto been unknown. We have established the first genetic system for the manipulation and production of a member of the non-classical succinate:quinone oxidoreductase family. Biochemical characterization of the W. succinogenes enzyme reveals that the putative SQR is in fact a novel methylmenaquinol:fumarate reductase (MFR) with no detectable succinate oxidation activity, clearly indicative of its involvement in anaerobic metabolism. We demonstrate that the hydrophilic subunits of the MFR complex are, in contrast to all other previously characterized members of the superfamily, exported into the periplasm via the twin-arginine translocation (tat)-pathway. Furthermore we show that a single amino acid exchange (Ala86-->His) in the flavoprotein of that enzyme complex is the only additional requirement for the covalent binding of the otherwise non-covalently bound FAD. Our results provide an explanation for the previously published puzzling observation that the C. jejuni sdhABE operon is upregulated in an oxygen-limited environment as compared with microaerophilic laboratory conditions.

The human Cdc37.Hsp90 complex studied by heteronuclear NMR spectroscopy.

The cell division cycle protein 37 (Cdc37) and the 90-kDa heat shock protein (Hsp90) are molecular chaperones, which are crucial elements in the protein signaling pathway. The largest class of client proteins for Cdc37 and Hsp90 are protein kinases. The catalytic domains of these kinases are stabilized by Cdc37, and their proper folding and functioning is dependent on Hsp90. Here, we present the x-ray crystal structure of the 16-kDa middle domain of human Cdc37 at 1.88 angstroms resolution and the structure of this domain in complex with the 23-kDa N-terminal domain of human Hsp90 based on heteronuclear solution state NMR data and docking. Our results demonstrate that the middle domain of Cdc37 exists as a monomer. NMR and mutagenesis experiments reveal Leu-205 in Cdc37 as a key residue enabling complex formation. These findings can be very useful in the development of small molecule inhibitors against cancer.