Yoshiaki Kawano

Crystal structures of the human adiponectin receptors

Adiponectin stimulation of its receptors, AdipoR1 and AdipoR2, increases the activities of 5′xa0AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR), respectively, thereby contributing to healthy longevity as key anti-diabetic molecules. AdipoR1 and AdipoR2 were predicted to contain seven transmembrane helices with the opposite topology to G-protein-coupled receptors. Here we report the crystal structures of human AdipoR1 and AdipoR2 at 2.9 and 2.4xa0Å resolution, respectively, which represent a novel class of receptor structure. The seven-transmembrane helices, conformationally distinct from those of G-protein-coupled receptors, enclose a large cavity where three conserved histidine residues coordinate a zinc ion. The zinc-binding structure may have a role in the adiponectin-stimulated AMPK phosphorylation and UCP2 upregulation. Adiponectin may broadly interact with the extracellular face, rather thanxa0the carboxy-terminal tail, of the receptors. The present information will facilitate the understanding of novel structure–function relationships and the development and optimization of AdipoR agonists for the treatment of obesity-related diseases, such as type 2 diabetes.

Achievement of protein micro-crystallography at SPring-8 beamline BL32XU

A micro-focused X-ray beam with size ranging from 1 × 1 to 10 × 10 μm has been achieved at beamline BL32XU at SPring-8, Japan. Combining the available micro-beam with newly developed techniques has enabled efficient protein micro-crystallography.

Crystal structure of the histidine-containing phosphotransfer protein ZmHP2 from maize.

In higher plants, histidine‐aspartate phosphorelays (two‐component system) are involved in hormone signaling and stress responses. In these systems, histidine‐containing phosphotransfer (HPt) proteins mediate the signal transmission from sensory histidine kinases to response regulators, including integration of several signaling pathways or branching into different pathways. We have determined the crystal structure of a maize HPt protein, ZmHP2, at 2.2 Å resolution. ZmHP2 has six α‐helices with a four‐helix bundle at the C‐terminus, a feature commonly found in HPt domains. In ZmHP2, almost all of the conserved residues among plant HPt proteins surround this histidine, probably forming the docking interface for the receiver domain of histidine kinase or the response regulator. Arg102 of ZmHP2 is conserved as a basic residue in plant HPt proteins. In bacteria, it is replaced by glutamine or glutamate that form a hydrogen bond to Nδ atoms of the phospho‐accepting histidine. It may play a key role in the complex formation of ZmHP2 with receiver domains.

Structural Basis for Catalytic Activation of Thiocyanate Hydrolase Involving Metal-Ligated Cysteine Modification

Thiocyanate hydrolase (SCNase) is a member of a family of nitrile hydratase proteins, each of which contains a unique noncorrin cobalt center with two post-translationally modified cysteine ligands, cysteine-sulfenic acid or -sulfenate (Cys-SO(H)), and cysteine-sulfininate (Cys-SO(2)(-)), respectively. We have found that a partially matured recombinant SCNase was activated during storage. The crystal structures of SCNase before and after storage demonstrated that Cys-SO(2)(-) modification of gammaCys131 proceeded to completion prior to storage, while Cys-SO(H) modification of gammaCys133 occurred during storage. SCNase activity was suppressed when gammaCys133 was further oxidized to Cys-SO(2)(-). The correlation between the catalytic activity and the extent of the gammaCys133 modification indicates that the cysteine sulfenic acid modification of gammaCys133 is of primary importance in determining the activity of SCNase.

New micro-beam beamline at SPring-8, targeting at protein micro-crystallography

A new protein micro‐crystallography beamline BL32XU at SPring‐8 is under construction and scheduled to start operation in 2010. The beamline is designed to provide the stabilized and brilliant micro‐beam to collect high‐quality data from micro‐crystals. The beamline consists of a hybrid in‐vacuum undulator, a liquid‐nitrogen cooled double crystal monochromator, and K‐B focusing mirrors with large magnification factor. Development of data acquisition system and end station consists of high‐precision diffractometer, high‐efficiency area detector, sample auto‐changer etc. are also in progress.

The bio-crystallography beamline (BL41XU) at SPring-8

The bio-crystallography beamline (BL41XU), one of two pilot beamlines at SPring-8, was constructed using a standard in-vacuum-type undulator and opened for general users from domestic and overseas countries. Many tests and improvements were carried out on beamline elements and equipment for macromolecular crystallography, especially on the so-called ‘‘pin-post’’ water cooling crystal of rotated-inclined double crystal monochromator. The maximum brilliance at sample position reached to 4 10 15 photons/s/mm 2 /mrad 2 at an X-ray energy of 11 keV. Commercially available X-ray detectors of CCD and imaging plate were installed in the experimental station. A beamline control software system for beam tracking and an on-line reader for large-format imaging plate were newly developed. # 2001 Elsevier Science B.V. All rights reserved. PACS: 61.10.Nz

FUNDAMENTAL DESIGN OF THE HIGH ENERGY UNDULATOR PILOT BEAMLINE FOR MACROMOLECULAR CRYSTALLOGRAPHY AT THE SPRING-8

The SPring‐8 project team adopted our proposal for public beamline as one of two pilot beamlines for standardizing the beamline elements. The light source of this beamline is an in‐vacuum‐type undulator of magnetic periodicity of 3.2 cm, which emits highly brilliant x rays in a wide energy range between 9 and 38 keV. The highest power emitted from the light source is 5 kW. The corresponding power density is 300 kW/mrad2. To handle the tremendous power density, grazing incidence diffraction with variable glancing angle will be used for the first crystal of a fixed‐exit double‐crystal monochromator. To focus the high energy x rays up to 38 keV, two supermirrors (Ovonic Synthetic Materials Co.) will be installed in vertical and horizontal directions independently to get a quasi‐isotropic and small beam profile at a focal position. In the experimental station, the sample crystals will be mounted on the κ‐type goniostat, and the Weissenberg photographs will be recorded by on‐line mode on an imaging plate detec...

Expression, purification, crystallization, and preliminary X-ray crystallographic studies of the human adiponectin receptors, AdipoR1 and AdipoR2

The adiponectin receptors (AdipoR1 and AdipoR2) are membrane proteins with seven transmembrane helices. These receptors regulate glucose and fatty acid metabolism, thereby ameliorating type 2 diabetes. The full-length human AdipoR1 and a series of N-terminally truncated mutants of human AdipoR1 and AdipoR2 were expressed in insect cells. In small-scale size exclusion chromatography, the truncated mutants AdipoR1Δ88 (residues 89–375) and AdipoR2Δ99 (residues 100–386) eluted mostly in the intact monodisperse state, while the others eluted primarily as aggregates. However, gel filtration chromatography of the large-scale preparation of the tag-affinity-purified AdipoR1Δ88 revealed the presence of an excessive amount of the aggregated state over the intact state. Since aggregation due to contaminating nucleic acids may have occurred during the sample concentration step, anion-exchange column chromatography was performed immediately after affinity chromatography, to separate the intact AdipoR1Δ88 from the aggregating species. The separated intact AdipoR1Δ88 did not undergo further aggregation, and was successfully purified to homogeneity by gel filtration chromatography. The purified AdipoR1Δ88 and AdipoR2Δ99 proteins were characterized by thermostability assays with 7-diethylamino-3-(4-maleimidophenyl)-4-methyl coumarin, thin layer chromatography of bound lipids, and surface plasmon resonance analysis of ligand binding, demonstrating their structural integrities. The AdipoR1Δ88 and AdipoR2Δ99 proteins were crystallized with the anti-AdipoR1 monoclonal antibody Fv fragment, by the lipidic mesophase method. X-ray diffraction data sets were obtained at resolutions of 2.8 and 2.4xa0Å, respectively.

Structural insights into the substrate specificity of bacterial copper amine oxidase obtained by using irreversible inhibitors.

Copper amine oxidases (CAOs) catalyse the oxidation of various aliphatic amines to the corresponding aldehydes, ammonia and hydrogen peroxide. Although CAOs from various organisms share a highly conserved active-site structure including a protein-derived cofactor, topa quinone (TPQ), their substrate specificities differ considerably. To obtain structural insights into the substrate specificity of a CAO from Arthrobacter globiformis (AGAO), we have determined the X-ray crystal structures of AGAO complexed with irreversible inhibitors that form covalent adducts with TPQ. Three hydrazine derivatives, benzylhydrazine (BHZ), 4-hydroxybenzylhydrazine (4-OH-BHZ) and phenylhydrazine (PHZ) formed predominantly a hydrazone adduct, which is structurally analogous to the substrate Schiff base of TPQ formed during the catalytic reaction. With BHZ and 4-OH-BHZ, but not with PHZ, the inhibitor aromatic ring is bound to a hydrophobic cavity near the active site in a well-defined conformation. Furthermore, the hydrogen atom on the hydrazone nitrogen is located closer to the catalytic base in the BHZ and 4-OH-BHZ adducts than in the PHZ adduct. These results correlate well with the reactivity of 2-phenylethylamine and tyramine as preferred substrates for AGAO and also explain why benzylamine is a poor substrate with markedly decreased rate constants for the steps of proton abstraction and the following hydrolysis.

Mutation of the N-terminal proline 9 of BLMA from Streptomyces verticillus abolishes the binding affinity for bleomycin

A gene, blmA, from bleomycin (Bm)‐producing Streptomyces verticillus, encodes a Bm‐binding protein, designated BLMA. The expression of BLMA conferred resistance to Bm in the Escherichia coli host, whereas a mutant protein, designated Pro‐9/Leu, with the N‐terminal proline 9 residue in BLMA replaced by leucine, did not. We created a fusion protein between the maltose‐binding protein (MBP) and a mutant protein Pro‐9/Leu/Leu with Met‐94 in Pro‐9/Leu replaced by leucine. Pro‐9/Leu/Leu from the fusion protein, obtained by digestion with CNBr digestion, did not inhibit DNA‐cleaving and antibacterial activities of Bm. Native‐polyacrylamide gel electrophoresis (PAGE) and gel filtration column chromatographic analysis showed that the molecular size of Pro‐9/Leu/Leu is roughly half of that of BLMA, suggesting that the mutant protein cannot form dimeric structure. Furthermore, Far‐UV circular dichroism (CD) spectrum of Pro‐9/Leu/Leu was quite different from that of BLMA and similar to the spectra obtained from unordered proteins [Venyaminov, S.Y. and Vassilenko, K.S. (1994) Anal. Biochem. 222, 176–184], suggesting that the secondary structure of Pro‐9/Leu/Leu is disrupted. These results indicate that the mutation abolishes not only dimer formation but also the secondary structure of BLMA, which results in the loss of its function as a Bm‐resistance determinant.