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.

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.

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.

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.

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 insight into substrate differentiation of the sugar-metabolizing enzyme galactitol dehydrogenase from Rhodobacter sphaeroides D.

Galactitol 2-dehydrogenase (GatDH) belongs to the protein superfamily of short-chain dehydrogenases. As an enzyme capable of the stereo- and regioselective modification of carbohydrates, it exhibits a high potential for application in biotechnology as a biocatalyst. We have determined the crystal structure of the binary form of GatDH in complex with its cofactor NAD(H) and of the ternary form in complex with NAD(H) and three different substrates. The active form of GatDH constitutes a homo-tetramer with two magnesium-ion binding sites each formed by two opposing C termini. The catalytic tetrad is formed by Asn(116), Ser(144), Tyr(159), and Lys(163). GatDH structurally aligns well with related members of the short-chain dehydrogenase family. The substrate binding pocket can be divided into two parts of different size and polarity. In the smaller part, the side chains of amino acids Ser(144), Ser(146), and Asn(151) are important determinants for the binding specificity and the orientation of (pro-) chiral compounds. The larger part of the pocket is elongated and flanked by polar and non-polar residues, enabling a rather broad substrate spectrum. The presented structures provide valuable information for a rational design of this enzyme to improve its stability against pH, temperature, or solvent concentration and to optimize product yield in bioreactors.

Structures of the nucleotide-binding domain of the human ABCB6 transporter and its complexes with nucleotides.

The human ATP-binding cassette (ABC) transporter ABCB6 is involved in haem-precursor transport across the mitochondrial membrane. The crystal structure of its nucleotide-binding domain (NBD) has been determined in the apo form and in complexes with ADP, with ADP and Mg(2+) and with ATP at high resolution. The overall structure is L-shaped and consists of two lobes, consistent with other reported NBD structures. Nucleotide binding is mediated by the highly conserved Tyr599 and the Walker A motif, and induces notable structural changes. Structural comparison with other structurally characterized NBDs and full-length ABC transporters gives the first insight into the possible catalytic mechanism of ABCB6 and the role of the N-terminal helix alpha(1) in full-length ABCB6.

The structure of the periplasmic thiol-disulfide oxidoreductase SoxS from Paracoccus pantotrophus indicates a triple Trx/Grx/DsbC functionality in chemotrophic sulfur oxidation.

The periplasmic thiol-disulfide oxidoreductase SoxS is beneficial for the sulfur-oxidizing (Sox) phenotype of the facultative chemotrophic bacterium Paracoccus pantotrophus and is not part of the Sox enzyme system. SoxS combines features of thioredoxins, glutaredoxins and the thiol-disulfide oxidoreductases of the Dsb family in structure, target specificity and reaction. The structure of SoxS was solved in oxidized and reduced forms at 2.1 and 1.9 A resolution, respectively. SoxS revealed high structural homology to typical cytoplasmic bacterial thioredoxins. In contrast, SoxS contained the active-site motif Pro-Gly-Cys-Leu-Tyr-Cys that is not present in other thioredoxins. Interestingly, the sequence of this motif is closely related to the Pro-Gly-Cys-Pro-Tyr-Cys sequence of some glutaredoxins and to the Pro-Xaa-Cys-Xaa-Tyr-Cys sequences of some members of the DsbC and DsbG subfamilies of thiol-disulfide oxidoreductases. Furthermore, the proposed substrate of SoxS, the interprotein disulfide of SoxY, Cys110(Y)-Cys110(Y), is structurally similar to oxidized glutathione. However, SoxS is proposed to specifically reduce the interprotein disulfide between two SoxY subunits, releasing a heterodimeric SoxYZ as an active part of the sulfur-oxidation cycle.

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.

Structural characterization of the thermostable Bradyrhizobium japonicumD-sorbitol dehydrogenase.

Bradyrhizobium japonicum sorbitol dehydrogenase is NADH-dependent and is active at elevated temperatures. The best substrate is D-glucitol (a synonym for D-sorbitol), although L-glucitol is also accepted, giving it particular potential in industrial applications. Crystallization led to a hexagonal crystal form, with crystals diffracting to 2.9 Šresolution. In attempts to phase the data, a molecular-replacement solution based upon PDB entry 4nbu (33% identical in sequence to the target) was found. The solution contained one molecule in the asymmetric unit, but a tetramer similar to that found in other short-chain dehydrogenases, including the search model, could be reconstructed by applying crystallographic symmetry operations. The active site contains electron density consistent with D-glucitol and phosphate, but there was not clear evidence for the binding of NADH. In a search for the features that determine the thermostability of the enzyme, the Tm for the orthologue from Rhodobacter sphaeroides, for which the structure was already known, was also determined, and this enzyme proved to be considerably less thermostable. A continuous ß-sheet is formed between two monomers in the tetramer of the B. japonicum enzyme, a feature not generally shared by short-chain dehydrogenases, and which may contribute to thermostability, as may an increased Pro/Gly ratio.

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.

Structural basis for complex formation between human IRSp53 and the translocated intimin receptor Tir of enterohemorrhagic E. coli.

Actin assembly beneath enterohemorrhagic E. coli (EHEC) attached to its host cell is triggered by the intracellular interaction of its translocated effector proteins Tir and EspF(U) with human IRSp53 family proteins and N-WASP. Here, we report the structure of the N-terminal I-BAR domain of IRSp53 in complex with a Tir-derived peptide, in which the homodimeric I-BAR domain binds two Tir molecules aligned in parallel. This arrangement provides a protein scaffold linking the bacterium to the host cell's actin polymerization machinery. The structure uncovers a specific peptide-binding site on the I-BAR surface, conserved between IRSp53 and IRTKS. The Tir Asn-Pro-Tyr (NPY) motif, essential for pedestal formation, is specifically recognized by this binding site. The site was confirmed by mutagenesis and in vivo-binding assays. It is possible that IRSp53 utilizes the NPY-binding site for additional interactions with as yet unknown partners within the host cell.

Viral preprotoxin signal sequence allows efficient secretion of green fluorescent protein by Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe.

Besides its importance as model organism in eukaryotic cell biology, yeast species have also developed into an attractive host for the expression, processing, and secretion of recombinant proteins. Here we investigated foreign protein secretion in four distantly related yeasts (Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe) by using green fluorescent protein (GFP) as a reporter and a viral secretion signal sequence derived from the K28 preprotoxin (pptox), the precursor of the yeast K28 virus toxin. In vivo expression of GFP fused to the N-terminal pptox leader sequence and/or expression of the entire pptox gene was driven either from constitutive (PGK1 and TPI1) or from inducible and/or repressible (GAL1, AOX1, and NMT1) yeast promoters. In each case, GFP entered the secretory pathway of the corresponding host cell; confocal fluorescence microscopy as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of cell-free culture supernatants confirmed that GFP was efficiently secreted into the culture medium. In addition to the results seen with GFP, the full-length viral pptox was correctly processed in all four yeast genera, leading to the secretion of a biologically active virus toxin. Taken together, our data indicate that the viral K28 pptox signal sequence has the potential for being used as a unique tool in recombinant protein production to ensure efficient protein secretion in yeast.