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52 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4, 5, 6 [Next/Last]

Instruments / Technical Area	Publication Details	Published
Krios I-Titan Krios I at Diamond	Molecular mechanism of SbmA, a promiscuous transporter exploited by antimicrobial peptides Dmitry Ghilarov , Satomi Inaba-Inoue , Piotr Stepien , Feng Qu , Elizabeth Michalczyk , Zuzanna Pakosz , Norimichi Nomura , Satoshi Ogasawara , Graham Charles Walker , Sylvie Rebuffat , So Iwata , Jonathan Gardiner Heddle , Konstantinos Beis Science Advances 7 DOI: 10.1126/sciadv.abj5363 Diamond Proposal Number(s): [18659] Open Access Show Abstract ▼ Abstract: Antibiotic metabolites and antimicrobial peptides mediate competition between bacterial species. Many of them hijack inner and outer membrane proteins to enter cells. Sensitivity of enteric bacteria to multiple peptide antibiotics is controlled by the single inner membrane protein SbmA. To establish the molecular mechanism of peptide transport by SbmA and related BacA, we determined their cryo–electron microscopy structures at 3.2 and 6 Å local resolution, respectively. The structures show a previously unknown fold, defining a new class of secondary transporters named SbmA-like peptide transporters. The core domain includes conserved glutamates, which provide a pathway for proton translocation, powering transport. The structures show an outward-open conformation with a large cavity that can accommodate diverse substrates. We propose a molecular mechanism for antibacterial peptide uptake paving the way for creation of narrow-targeted therapeutics.	Sep 2021
	Ubiquinone binding site of yeast NADH dehydrogenase revealed by structures binding novel competitive- and mixed-type inhibitors Tetsuo Yamashita , Daniel Ken Inaoka , Tomoo Shiba , Takumi Oohashi , So Iwata , Takao Yagi , Hiroaki Kosaka , Hideto Miyoshi , Shigeharu Harada , Kiyoshi Kita , Katsuya Hirano Scientific Reports 8 DOI: 10.1038/s41598-018-20775-6 Open Access Show Abstract ▼ Abstract: Yeast Ndi1 is a monotopic alternative NADH dehydrogenase. Its crystal structure in complex with the electron acceptor, ubiquinone, has been determined. However, there has been controversy regarding the ubiquinone binding site. To address these points, we identified the first competitive inhibitor of Ndi1, stigmatellin, along with new mixed-type inhibitors, AC0-12 and myxothiazol, and thereby determined the crystal structures of Ndi1 in complexes with the inhibitors. Two separate binding sites of stigmatellin, STG-1 and STG-2, were observed. The electron density at STG-1, located at the vicinity of the FAD cofactor, further demonstrated two binding modes: STG-1a and STG-1b. AC0-12 and myxothiazol are also located at the vicinity of FAD. The comparison of the binding modes among stigmatellin at STG-1, AC0-12, and myxothiazol revealed a unique position for the aliphatic tail of stigmatellin at STG-1a. Mutations of amino acid residues that interact with this aliphatic tail at STG-1a reduced the affinity of Ndi1 for ubiquinone. In conclusion, the position of the aliphatic tail of stigmatellin at STG-1a provides a structural basis for its competitive inhibition of Ndi1. The inherent binding site of ubiquinone is suggested to overlap with STG-1a that is distinct from the binding site for NADH.	Feb 2018
B22-Multimode InfraRed imaging And Microspectroscopy I03-Macromolecular Crystallography I04-Macromolecular Crystallography	X-ray free electron laser determination of crystal structures of dark and light states of a reversibly photoswitching fluorescent protein at room temperature Christopher D. M. Hutchison , Violeta Cordon-Preciado , Rhodri M. L. Morgan , Takanori Nakane , Josie Ferreira , Gabriel Dorlhiac , Alvaro Sanchez-Gonzalez , Allan S. Johnson , Ann Fitzpatrick , Clyde Fare , Jon Marangos , Chun Hong Yoon , Mark S. Hunter , Daniel P. Deponte , Sébastien Boutet , Shigeki Owada , Rie Tanaka , Kensuke Tono , So Iwata , Jasper J. Van Thor International Journal Of Molecular Sciences 18 DOI: 10.3390/ijms18091918 Diamond Proposal Number(s): [12579] Open Access Show Abstract ▼ Abstract: The photochromic fluorescent protein Skylan-NS (Nonlinear Structured illumination variant mEos3.1H62L) is a reversibly photoswitchable fluorescent protein which has an unilluminated/ground state with an anionic and cis chromophore conformation and high fluorescence quantum yield. Photo-conversion with illumination at 515 nm generates a meta-stable intermediate with neutral trans-chromophore structure that has a 4 h lifetime. We present X-ray crystal structures of the cis (on) state at 1.9 Angstrom resolution and the trans (off) state at a limiting resolution of 1.55 Angstrom from serial femtosecond crystallography experiments conducted at SPring-8 Angstrom Compact Free Electron Laser (SACLA) at 7.0 keV and 10.5 keV, and at Linac Coherent Light Source (LCLS) at 9.5 keV. We present a comparison of the data reduction and structure determination statistics for the two facilities which differ in flux, beam characteristics and detector technologies. Furthermore, a comparison of droplet on demand, grease injection and Gas Dynamic Virtual Nozzle (GDVN) injection shows no significant differences in limiting resolution. The photoconversion of the on- to the off-state includes both internal and surface exposed protein structural changes, occurring in regions that lack crystal contacts in the orthorhombic crystal form.	Sep 2017
I04-Macromolecular Crystallography	Structure of eukaryotic purine/H+ symporter UapA suggests a role for homodimerization in transport activity Yilmaz Alguel , Sotiris Amillis , James Leung , George Lambrinidis , Stefano Capaldi , Nicola J. Scull , Gregory Craven , So Iwata , Alan Armstrong , Emmanuel Mikros , George Diallinas , Alexander D. Cameron , Bernadette Byrne Nature Communications 7 DOI: 10.1038/ncomms11336 Open Access Show Abstract ▼ Abstract: The uric acid/xanthine H+ symporter, UapA, is a high-affinity purine transporter from the filamentous fungus Aspergillus nidulans. Here we present the crystal structure of a genetically stabilized version of UapA (UapA-G411VΔ1–11) in complex with xanthine. UapA is formed from two domains, a core domain and a gate domain, similar to the previously solved uracil transporter UraA, which belongs to the same family. The structure shows UapA in an inward-facing conformation with xanthine bound to residues in the core domain. Unlike UraA, which was observed to be a monomer, UapA forms a dimer in the crystals with dimer interactions formed exclusively through the gate domain. Analysis of dominant negative mutants is consistent with dimerization playing a key role in transport. We postulate that UapA uses an elevator transport mechanism likely to be shared with other structurally homologous transporters including anion exchangers and prestin.	Apr 2016
I04-1-Macromolecular Crystallography (fixed wavelength)	Crystal structure of the anion exchanger domain of human erythrocyte band 3 T. Arakawa , T. Kobayashi-Yurugi , Y. Alguel , H. Iwanari , H. Hatae , M. Iwata , Y. Abe , T. Hino , C. Ikeda-Suno , H. Kuma , D. Kang , T. Murata , T. Hamakubo , A. D. Cameron , T. Kobayashi , N. Hamasaki , S. Iwata Science 350 (6261), 680 - 684 DOI: 10.1126/science.aaa4335 PMID: 26542571 Show Abstract ▼ Abstract: Anion exchanger 1 (AE1), also known as band 3 or SLC4A1, plays a key role in the removal of carbon dioxide from tissues by facilitating the exchange of chloride and bicarbonate across the plasma membrane of erythrocytes. An isoform of AE1 is also present in the kidney. Specific mutations in human AE1 cause several types of hereditary hemolytic anemias and/or distal renal tubular acidosis. Here we report the crystal structure of the band 3 anion exchanger domain (AE1CTD) at 3.5 angstroms. The structure is locked in an outward-facing open conformation by an inhibitor. Comparing this structure with a substrate-bound structure of the uracil transporter UraA in an inward-facing conformation allowed us to identify the anion-binding position in the AE1CTD, and to propose a possible transport mechanism that could explain why selected mutations lead to disease.	Nov 2015
I02-Macromolecular Crystallography	Structure and mechanism of the mammalian fructose transporter GLUT5 Norimichi Nomura , Gregory Verdon , Haejoo Kang , Tatsuro Shimamura , Yayoi Nomura , Yo Sonoda , Saba Abdul Hussein , Aziz Qureshi , Mathieu Coincon , Yumi Sato , Hitomi Abe , Yoshiko Nakada-Nakura , Tomoya Hino , Takatoshi Arakawa , Osamu Kusano-Arai , Hiroko Iwanari , Takeshi Murata , Takuya Kobayashi , Takao Hamakubo , Michihiro Kasahara , So Iwata , David Drew Nature 526, 397 - 401 DOI: 10.1038/nature14909 PMID: 26416735 Show Abstract ▼ Abstract: The altered activity of the fructose transporter GLUT5, an isoform of the facilitated-diffusion glucose transporter family, has been linked to disorders such as type 2 diabetes and obesity. GLUT5 is also overexpressed in certain tumour cells, and inhibitors are potential drugs for these conditions. Here we describe the crystal structures of GLUT5 from Rattus norvegicus and Bos taurus in open outward- and open inward-facing conformations, respectively. GLUT5 has a major facilitator superfamily fold like other homologous monosaccharide transporters. On the basis of a comparison of the inward-facing structures of GLUT5 and human GLUT1, a ubiquitous glucose transporter, we show that a single point mutation is enough to switch the substrate-binding preference of GLUT5 from fructose to glucose. A comparison of the substrate-free structures of GLUT5 with occluded substrate-bound structures of Escherichia coli XylE suggests that, in addition to global rocker-switch-like re-orientation of the bundles, local asymmetric rearrangements of carboxy-terminal transmembrane bundle helices TM7 and TM10 underlie a ‘gated-pore’ transport mechanism in such monosaccharide transporters.	Oct 2015
I24-Microfocus Macromolecular Crystallography	Structure determination of an integral membrane protein at room temperature from crystals in situ Danny Axford , James Foadi , Nien-Jen Hu , Hassan Choudhury , So Iwata , Konstantinos Beis , Gwyndaf Evans , Yilmaz Alguel Acta Crystallographica Section D Biological Crystallography 71 DOI: 10.1107/S139900471500423X PMID: 26057664 Open Access Show Abstract ▼ Abstract: The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 A˚ resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third generation synchrotron beamlines.	Jun 2015
I24-Microfocus Macromolecular Crystallography	Crystal structures of the human adiponectin receptors Hiroaki Tanabe , Yoshifumi Fujii , Miki Okada-Iwabu , Masato Iwabu , Yoshihiro Nakamura , Toshiaki Hosaka , Kanna Motoyama , Mariko Ikeda , Motoaki Wakiyama , Takaho Terada , Noboru Ohsawa , Masakatsu Hato , Satoshi Ogasawara , Tomoya Hino , Takeshi Murata , So Iwata , Kunio Hirata , Yoshiaki Kawano , Masaki Yamamoto , Tomomi Kimura-Someya Nature 520, 312 - 316 DOI: 10.1038/nature14301 PMID: 25855295 Show Abstract ▼ Abstract: Adiponectin stimulation of its receptors, AdipoR1 and AdipoR2, increases the activities of 5′ AMP-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 2.4 Å 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 than the 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.	Apr 2015
I24-Microfocus Macromolecular Crystallography	Expression, purification, crystallization, and preliminary X-ray crystallographic studies of the human adiponectin receptors, AdipoR1 and AdipoR2 Hiroaki Tanabe , Kanna Motoyama , Mariko Ikeda , Motoaki Wakiyama , Takaho Terada , Noboru Ohsawa , Toshiaki Hosaka , Masakatsu Hato , Yoshifumi Fujii , Yoshihiro Nakamura , Satoshi Ogasawara , Tomoya Hino , Takeshi Murata , So Iwata , Miki Okada-Iwabu , Masato Iwabu , Kunio Hirata , Yoshiaki Kawano , Masaki Yamamoto , Tomomi Kimura-Someya , Mikako Shirouzu , Toshimasa Yamauchi , Takashi Kadowaki , Shigeyuki Yokoyama Journal Of Structural And Functional Genomics DOI: 10.1007/s10969-014-9192-z Open Access Show Abstract ▼ Abstract: 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 2.4 Å, respectively.	Jan 2015
I24-Microfocus Macromolecular Crystallography	Crystal structure of the sodium-proton antiporter NhaA dimer and new mechanistic insights C. Lee , S. Yashiro , D. L. Dotson , P. Uzdavinys , S. Iwata , M. S. P. Sansom , C. Von Ballmoos , O. Beckstein , D. Drew , A. D. Cameron The Journal Of General Physiology 144 (6), 529 - 544 DOI: 10.1085/jgp.201411219 PMID: 25422503 Open Access Show Abstract ▼ Abstract: Sodium–proton antiporters rapidly exchange protons and sodium ions across the membrane to regulate intracellular pH, cell volume, and sodium concentration. How ion binding and release is coupled to the conformational changes associated with transport is not clear. Here, we report a crystal form of the prototypical sodium–proton antiporter NhaA from Escherichia coli in which the protein is seen as a dimer. In this new structure, we observe a salt bridge between an essential aspartic acid (Asp163) and a conserved lysine (Lys300). An equivalent salt bridge is present in the homologous transporter NapA, but not in the only other known crystal structure of NhaA, which provides the foundation of most existing structural models of electrogenic sodium–proton antiport. Molecular dynamics simulations show that the stability of the salt bridge is weakened by sodium ions binding to Asp164 and the neighboring Asp163. This suggests that the transport mechanism involves Asp163 switching between forming a salt bridge with Lys300 and interacting with the sodium ion. pKa calculations suggest that Asp163 is highly unlikely to be protonated when involved in the salt bridge. As it has been previously suggested that Asp163 is one of the two residues through which proton transport occurs, these results have clear implications to the current mechanistic models of sodium–proton antiport in NhaA.	Nov 2014

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