Soichi Wakatsuki

All Publications

Environmental Calcium Controls Alternate Physical States of the Caulobacter Surface Layer BIOPHYSICAL JOURNAL Herrmann, J., Jabbarpour, F., Bargar, P. G., Nomellini, J. F., Li, P., Lane, T. J., Weiss, T. M., Smit, J., Shapiro, L., Wakatsuki, S. 2017; 112 (9): 1841-1851
Abstract

Surface layers (S-layers) are paracrystalline, proteinaceous structures found in most archaea and many bacteria. Often the outermost cell envelope component, S-layers serve diverse functions including aiding pathogenicity and protecting against predators. We report that the S-layer of Caulobacter crescentus exhibits calcium-mediated structural plasticity, switching irreversibly between an amorphous aggregate state and the crystalline state. This finding invalidates the common assumption that S-layers serve only as static wall-like structures. In vitro, the Caulobacter S-layer protein, RsaA, enters the aggregate state at physiological temperatures and low divalent calcium ion concentrations. At higher concentrations, calcium ions stabilize monomeric RsaA, which can then transition to the two-dimensional crystalline state. Caulobacter requires micromolar concentrations of calcium for normal growth and development. Without an S-layer, Caulobacter is even more sensitive to changes in environmental calcium concentration. Therefore, this structurally dynamic S-layer responds to environmental conditions as an ion sensor and protects Caulobacter from calcium deficiency stress, a unique mechanism of bacterial adaptation. These findings provide a biochemical and physiological basis for RsaA's calcium-binding behavior, which extends far beyond calcium's commonly accepted role in aiding S-layer biogenesis or oligomerization and demonstrates a connection to cellular fitness.

View details for DOI 10.1016/j.bpj.2017.04.003

View details for Web of Science ID 000401301600013

View details for PubMedID 28494955
Phosphorylation of the mitochondrial autophagy receptor Nix enhances its interaction with LC3 proteins SCIENTIFIC REPORTS Rogov, V. V., Suzuki, H., Marinkovic, M., Lang, V., Kato, R., Kawasaki, M., Buljubasic, M., Sprung, M., Rogova, N., Wakatsuki, S., Hamacher-Brady, A., Doetsch, V., Dikic, I., Brady, N. R., Novak, I. 2017; 7
Abstract

The mitophagy receptor Nix interacts with LC3/GABARAP proteins, targeting mitochondria into autophagosomes for degradation. Here we present evidence for phosphorylation-driven regulation of the Nix:LC3B interaction. Isothermal titration calorimetry and NMR indicate a ~100 fold enhanced affinity of the serine 34/35-phosphorylated Nix LC3-interacting region (LIR) to LC3B and formation of a very rigid complex compared to the non-phosphorylated sequence. Moreover, the crystal structure of LC3B in complex with the Nix LIR peptide containing glutamic acids as phosphomimetic residues and NMR experiments revealed that LIR phosphorylation stabilizes the Nix:LC3B complex via formation of two additional hydrogen bonds between phosphorylated serines of Nix LIR and Arg11, Lys49 and Lys51 in LC3B. Substitution of Lys51 to Ala in LC3B abrogates binding of a phosphomimetic Nix mutant. Functionally, serine 34/35 phosphorylation enhances autophagosome recruitment to mitochondria in HeLa cells. Together, this study provides cellular, biochemical and biophysical evidence that phosphorylation of the LIR domain of Nix enhances mitophagy receptor engagement.

View details for DOI 10.1038/s41598-017-01258-6

View details for Web of Science ID 000400104200020

View details for PubMedID 28442745
Se-SAD serial femtosecond crystallography datasets from selenobiotinyl-streptavidin SCIENTIFIC DATA Yoon, C. H., Demirci, H., Sierra, R. G., Dao, E. H., Ahmadi, R., Aksit, F., Aquila, A. L., Batyuk, A., Ciftci, H., Guillet, S., Hayes, M. J., Hayes, B., Lane, T. J., Liang, M., Lundstrom, U., Koglin, J. E., Mgbam, P., Rao, Y., Rendahl, T., Rodriguez, E., Zhang, L., Wakatsuki, S., Boutet, S., Holton, J. M., Hunter, M. S. 2017; 4
Abstract

We provide a detailed description of selenobiotinyl-streptavidin (Se-B SA) co-crystal datasets recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS) for selenium single-wavelength anomalous diffraction (Se-SAD) structure determination. Se-B SA was chosen as the model system for its high affinity between biotin and streptavidin where the sulfur atom in the biotin molecule (C10H16N2O3S) is substituted with selenium. The dataset was collected at three different transmissions (100, 50, and 10%) using a serial sample chamber setup which allows for two sample chambers, a front chamber and a back chamber, to operate simultaneously. Diffraction patterns from Se-B SA were recorded to a resolution of 1.9 Å. The dataset is publicly available through the Coherent X-ray Imaging Data Bank (CXIDB) and also on LCLS compute nodes as a resource for research and algorithm development.

View details for DOI 10.1038/sdata.2017.55

View details for Web of Science ID 000400151700004

View details for PubMedID 28440794
Responses to 'Atomic resolution': a badly abused term in structural biology ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY Chiu, W., Holton, J., Langan, P., Sauter, N. K., Schlichting, I., Terwilliger, T., Martin, J. L., Read, R. J., Wakatsuki, S. 2017; 73: 381-383
View details for DOI 10.1107/S205979831700417X

View details for Web of Science ID 000398536800010

View details for PubMedID 28375150
Structural and functional analysis of the GABARAP interaction motif (GIM). EMBO reports Rogov, V. V., Stolz, A., Ravichandran, A. C., Rios-Szwed, D. O., Suzuki, H., Kniss, A., Löhr, F., Wakatsuki, S., Dötsch, V., Dikic, I., Dobson, R. C., McEwan, D. G. 2017; 18 (8): 1382–96
Abstract

Through the canonical LC3 interaction motif (LIR), [W/F/Y]-X1-X2-[I/L/V], protein complexes are recruited to autophagosomes to perform their functions as either autophagy adaptors or receptors. How these adaptors/receptors selectively interact with either LC3 or GABARAP families remains unclear. Herein, we determine the range of selectivity of 30 known core LIR motifs towards individual LC3s and GABARAPs. From these, we define a G ABARAP I nteraction M otif (GIM) sequence ([W/F]-[V/I]-X2-V) that the adaptor protein PLEKHM1 tightly conforms to. Using biophysical and structural approaches, we show that the PLEKHM1-LIR is indeed 11-fold more specific for GABARAP than LC3B. Selective mutation of the X1and X2positions either completely abolished the interaction with all LC3 and GABARAPs or increased PLEKHM1-GIM selectivity 20-fold towards LC3B. Finally, we show that conversion of p62/SQSTM1, FUNDC1 and FIP200 LIRs into our newly defined GIM, by introducing two valine residues, enhances their interaction with endogenous GABARAP over LC3B. The identification of a GABARAP-specific interaction motif will aid the identification and characterization of the expanding array of autophagy receptor and adaptor proteins and theirin vivofunctions.

View details for DOI 10.15252/embr.201643587

View details for PubMedID 28655748

View details for PubMedCentralID PMC5538626
Integrated structural biology and molecular ecology of N-cycling enzymes from ammonia-oxidizing archaea. Environmental microbiology reports Tolar, B. B., Herrmann, J., Bargar, J. R., van den Bedem, H., Wakatsuki, S., Francis, C. A. 2017; 9 (5): 484–91
Abstract

Knowledge of the molecular ecology and environmental determinants of ammonia-oxidizing organisms is critical to understanding and predicting the global nitrogen (N) and carbon cycles, but an incomplete biochemical picture hinders in vitro studies of N-cycling enzymes. Although an integrative structural and dynamic characterization at the atomic scale would advance our understanding of function tremendously, structural knowledge of key N-cycling enzymes from ecologically relevant ammonia oxidizers is unfortunately extremely limited. Here, we discuss the challenges and opportunities for examining the ecology of ammonia-oxidizing organisms, particularly uncultivated Thaumarchaeota, through (meta)genome-driven structural biology of the enzymes ammonia monooxygenase (AMO) and nitrite reductase (NirK).

View details for DOI 10.1111/1758-2229.12567

View details for PubMedID 28677304
Selenium single-wavelength anomalous diffraction de novo phasing using an X-ray-free electron laser. Nature communications Hunter, M. S., Yoon, C. H., Demirci, H., Sierra, R. G., Dao, E. H., Ahmadi, R., Aksit, F., Aquila, A. L., Ciftci, H., Guillet, S., Hayes, M. J., Lane, T. J., Liang, M., Lundström, U., Koglin, J. E., Mgbam, P., Rao, Y., Zhang, L., Wakatsuki, S., Holton, J. M., Boutet, S. 2016; 7: 13388-?
Abstract

Structural information about biological macromolecules near the atomic scale provides important insight into the functions of these molecules. To date, X-ray crystallography has been the predominant method used for macromolecular structure determination. However, challenges exist when solving structures with X-rays, including the phase problem and radiation damage. X-ray-free electron lasers (X-ray FELs) have enabled collection of diffraction information before the onset of radiation damage, yet the majority of structures solved at X-ray FELs have been phased using external information via molecular replacement. De novo phasing at X-ray FELs has proven challenging due in part to per-pulse variations in intensity and wavelength. Here we report the solution of a selenobiotinyl-streptavidin structure using phases obtained by the anomalous diffraction of selenium measured at a single wavelength (Se-SAD) at the Linac Coherent Light Source. Our results demonstrate Se-SAD, routinely employed at synchrotrons for novel structure determination, is now possible at X-ray FELs.

View details for DOI 10.1038/ncomms13388

View details for PubMedID 27811937
A novel mode of ubiquitin recognition by the ubiquitin-binding zinc finger domain of WRNIP1 FEBS JOURNAL Suzuki, N., Rohaim, A., Kato, R., Dikic, I., Wakatsuki, S., Kawasaki, M. 2016; 283 (11): 2004-2017
Abstract

The ubiquitin-binding zinc finger (UBZ) is a type of zinc-coordinating β-β-α fold domain found mainly in proteins involved in DNA repair and transcriptional regulation. Here we report the crystal structure of the UBZ domain of Y-family DNA polymerase (pol) η and the crystal structure of the complex between the UBZ domain of Werner helicase-interacting protein 1 (WRNIP1) and ubiquitin, crystallized using the green fluorescent protein fusion technique. In contrast to the pol η UBZ, which has been proposed to bind ubiquitin via its C-terminal α helix, ubiquitin binds to a novel surface of WRNIP1 UBZ composed of the first β strand and the C-terminal α helix. In addition, we report the structure of the tandem UBZ domains of Tax1-binding protein 1 (TAX1BP1) and show that the second UBZ of TAX1BP1 binds ubiquitin, presumably in a manner similar to that of WRNIP1 UBZ. We propose that UBZ domains can be divided into at least two different types in terms of the ubiquitin-binding surfaces: the pol η type and the WRNIP1 type. This article is protected by copyright. All rights reserved.

View details for DOI 10.1111/febs.13734

View details for Web of Science ID 000381316600001

View details for PubMedID 27062441
Selective Binding of AIRAPL Tandem UIMs to Lys48-Linked Tri-Ubiquitin Chains STRUCTURE Rahighi, S., Braunstein, I., Ternette, N., Kessler, B., Kawasaki, M., Kato, R., Matsui, T., Weiss, T. M., Stanhill, A., Wakatsuki, S. 2016; 24 (3): 412-422
Abstract

Lys48-linked ubiquitin chains act as the main targeting signals for protein degradation by the proteasome. Here we report selective binding of AIRAPL, a protein that associates with the proteasome upon exposure to arsenite, to Lys48-linked tri-ubiquitin chains. AIRAPL comprises two ubiquitin-interacting motifs in tandem (tUIMs) that are linked through a flexible inter-UIM region. In the complex crystal structure UIM1 binds the proximal ubiquitin, whereas UIM2 (the double-sided UIM) binds non-symmetrically to the middle and distal ubiquitin moieties on either side of the helix. Specificity of AIRAPL for Lys48-linked ubiquitin chains is determined by UIM2, and the flexible inter-UIM linker increases avidity by placing the two UIMs in an orientation that facilitates binding of the third ubiquitin to UIM1. Unlike middle and proximal ubiquitins, distal ubiquitin binds UIM2 through a novel surface, which leaves the Ile44 hydrophobic patch accessible for binding to the proteasomal ubiquitin receptors.

View details for DOI 10.1016/j.str.2015.12.017

View details for Web of Science ID 000373568300009

View details for PubMedID 26876100
Expanding beyond biological crystallography. Acta crystallographica. Section D, Structural biology Martin, J. L., Read, R. J., Wakatsuki, S. 2016; 72: 1-?
View details for DOI 10.1107/S2059798315023761

View details for PubMedID 26894528
The linac coherent light source single particle imaging road map STRUCTURAL DYNAMICS Aquila, A., Barty, A., Bostedt, C., Boutet, S., Carini, G., Deponte, D., DRELL, P., Doniach, S., Downing, K. H., Earnest, T., Elmlund, H., Elser, V., Guehr, M., Hajdu, J., Hastings, J., Hau-Riege, S. P., Huang, Z., Lattman, E. E., Maia, F. R., Marchesini, S., Ourmazd, A., Pellegrini, C., Santra, R., Schlichting, I., Schroer, C., Spence, J. C., Vartanyants, I. A., Wakatsuki, S., Weis, W. I., Williams, G. J. 2015; 2 (4)
View details for DOI 10.1063/1.4918726

View details for Web of Science ID 000360649200003
Gliotoxin Suppresses NF-kappa B Activation by Selectively Inhibiting Linear Ubiquitin Chain Assembly Complex (LUBAC) ACS CHEMICAL BIOLOGY Sakamoto, H., Egashira, S., Saito, N., Kirisako, T., Miller, S., Sasaki, Y., Matsumoto, T., Shimonishi, M., Komatsu, T., Terai, T., Ueno, T., Hanaoka, K., Kojima, H., Okabe, T., Wakatsuki, S., Iwai, K., Nagano, T. 2015; 10 (3): 675-681
Abstract

A linear ubiquitin chain, which consists of ubiquitin molecules linked via their N- and C-termini, is formed by a linear ubiquitin chain assembly complex (LUBAC) composed of HOIP, HOIL-1L, and SHARPIN, and conjugation of a linear ubiquitin chain on the NF-κB essential modulator (NEMO) is deeply involved in NF-κB activation induced by various signals. Since abnormal activation of NF-κB is associated with inflammatory disease and malignancy, we searched for an inhibitor of LUBAC by high-throughput screening (HTS) with a Tb(3+)-fluorescein FRET system. As a result, we found that the fungal metabolite gliotoxin inhibits LUBAC selectively by binding to the RING-IBR-RING domain of HOIP, the catalytic center of LUBAC. Gliotoxin has been well-known as an inhibitor of NF-κB activation, though its action mechanism has remained elusive. Here, we show that gliotoxin inhibits signal-induced NF-κB activation by selectively inhibiting LUBAC-mediated linear ubiquitin chain formation.

View details for DOI 10.1021/cb500653y

View details for Web of Science ID 000351558700004

View details for PubMedID 25494483
High-intensity double-pulse X-ray free-electron laser NATURE COMMUNICATIONS Marinelli, A., Ratner, D., Lutman, A. A., Turner, J., Welch, J., Decker, F., Loos, H., BEHRENS, C., Gilevich, S., Miahnahri, A. A., Vetter, S., Maxwell, T. J., Ding, Y., Coffee, R., Wakatsuki, S., Huang, Z. 2015; 6
Abstract

The X-ray free-electron laser has opened a new era for photon science, improving the X-ray brightness by ten orders of magnitude over previously available sources. Similar to an optical laser, the spectral and temporal structure of the radiation pulses can be tailored to the specific needs of many experiments by accurately manipulating the lasing medium, that is, the electron beam. Here we report the generation of mJ-level two-colour hard X-ray pulses of few femtoseconds duration with an XFEL driven by twin electron bunches at the Linac Coherent Light Source. This performance represents an improvement of over an order of magnitude in peak power over state-of-the-art two-colour XFELs. The unprecedented intensity and temporal coherence of this new two-colour X-ray free-electron laser enable an entirely new set of scientific applications, ranging from X-ray pump/X-ray probe experiments to the imaging of complex biological samples with multiple wavelength anomalous dispersion.

View details for DOI 10.1038/ncomms7369

View details for Web of Science ID 000352692100003

View details for PubMedID 25744344
PLEKHM1 Regulates Autophagosome-Lysosome Fusion through HOPS Complex and LC3/GABARAP Proteins MOLECULAR CELL McEwan, D. G., Popovic, D., Gubas, A., Terawaki, S., Suzuki, H., Stadel, D., Coxon, F. P., de Stegmann, D. M., Bhogaraju, S., Maddi, K., Kirchof, A., Gatti, E., Helfrich, M. H., Wakatsuki, S., Behrends, C., Pierre, P., Dikic, I. 2015; 57 (1): 39-54
Abstract

The lysosome is the final destination for degradation of endocytic cargo, plasma membrane constituents, and intracellular components sequestered by macroautophagy. Fusion of endosomes and autophagosomes with the lysosome depends on the GTPase Rab7 and the homotypic fusion and protein sorting (HOPS) complex, but adaptor proteins that link endocytic and autophagy pathways with lysosomes are poorly characterized. Herein, we show that Pleckstrin homology domain containing protein family member 1 (PLEKHM1) directly interacts with HOPS complex and contains a LC3-interacting region (LIR) that mediates its binding to autophagosomal membranes. Depletion of PLEKHM1 blocks lysosomal degradation of endocytic (EGFR) cargo and enhances presentation of MHC class I molecules. Moreover, genetic loss of PLEKHM1 impedes autophagy flux upon mTOR inhibition and PLEKHM1 regulates clearance of protein aggregates in an autophagy- and LIR-dependent manner. PLEKHM1 is thus a multivalent endocytic adaptor involved in the lysosome fusion events controlling selective and nonselective autophagy pathways.

View details for DOI 10.1016/j.molcel.2014.11.006

View details for Web of Science ID 000347711100005

View details for PubMedID 25498145
Structural Analysis of the Complex between Penta-EF-Hand ALG-2 Protein and Sec31A Peptide Reveals a Novel Target Recognition Mechanism of ALG-2. International journal of molecular sciences Takahashi, T., Kojima, K., Zhang, W., Sasaki, K., Ito, M., Suzuki, H., Kawasaki, M., Wakatsuki, S., Takahara, T., Shibata, H., Maki, M. 2015; 16 (2): 3677-3699
Abstract

ALG-2, a 22-kDa penta-EF-hand protein, is involved in cell death, signal transduction, membrane trafficking, etc., by interacting with various proteins in mammalian cells in a Ca2+-dependent manner. Most known ALG-2-interacting proteins contain proline-rich regions in which either PPYPXnYP (type 1 motif) or PXPGF (type 2 motif) is commonly found. Previous X-ray crystal structural analysis of the complex between ALG-2 and an ALIX peptide revealed that the peptide binds to the two hydrophobic pockets. In the present study, we resolved the crystal structure of the complex between ALG-2 and a peptide of Sec31A (outer shell component of coat complex II, COPII; containing the type 2 motif) and found that the peptide binds to the third hydrophobic pocket (Pocket 3). While amino acid substitution of Phe85, a Pocket 3 residue, with Ala abrogated the interaction with Sec31A, it did not affect the interaction with ALIX. On the other hand, amino acid substitution of Tyr180, a Pocket 1 residue, with Ala caused loss of binding to ALIX, but maintained binding to Sec31A. We conclude that ALG-2 recognizes two types of motifs at different hydrophobic surfaces. Furthermore, based on the results of serial mutational analysis of the ALG-2-binding sites in Sec31A, the type 2 motif was newly defined.

View details for DOI 10.3390/ijms16023677

View details for PubMedID 25667979
Demonstration of Single-Crystal Self-Seeded Two-Color X-Ray Free-Electron Lasers PHYSICAL REVIEW LETTERS Lutman, A. A., Decker, F., ARTHUR, J., Chollet, M., Feng, Y., Hastings, J., Huang, Z., Lemke, H., Nuhn, H., Marinelli, A., Turner, J. L., Wakatsuki, S., Welch, J., Zhu, D. 2014; 113 (25)
Abstract

A scheme for generating two simultaneous hard-x-ray free-electron laser pulses with a controllable difference in photon energy is described and then demonstrated using the self-seeding setup at the Linac Coherent Light Source (LCLS). The scheme takes advantage of the existing LCLS equipment, which allows two independent rotations of the self-seeding diamond crystal. The two degrees of freedom are used to select two nearby crystal reflections, causing two wavelengths to be present in the forward transmitted seeding x-ray pulse. The free-electron laser system must support amplification at both desired wavelengths.

View details for DOI 10.1103/PhysRevLett.113.254801

View details for Web of Science ID 000349416600005

View details for PubMedID 25554887
Goniometer-based femtosecond crystallography with X-ray free electron lasers PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cohen, A. E., Soltis, S. M., Gonzalez, A., Aguila, L., Alonso-Mori, R., Barnes, C. O., Baxter, E. L., Brehmer, W., Brewster, A. S., Brunger, A. T., Calero, G., Chang, J. F., Chollet, M., Ehrensberger, P., Eriksson, T. L., Feng, Y., Hattne, J., Hedman, B., Hollenbeck, M., Holton, J. M., Keable, S., Kobilka, B. K., Kovaleva, E. G., Kruse, A. C., Lemke, H. T., Lin, G., Lyubimov, A. Y., Manglik, A., Mathews, I. I., McPhillips, S. E., Nelson, S., Peters, J. W., Sauter, N. K., Smith, C. A., Song, J., Stevenson, H. P., Tsai, Y., Uervirojnangkoorn, M., Vinetsky, V., Wakatsuki, S., Weis, W. I., Zadvornyy, O. A., Zeldin, O. B., Zhu, D., Hodgson, K. O. 2014; 111 (48): 17122-17127
Abstract

The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.

View details for DOI 10.1073/pnas.1418733111

View details for Web of Science ID 000345920800042

View details for PubMedID 25362050

View details for PubMedCentralID PMC4260607
Mechanism Underlying I kappa B Kinase Activation Mediated by the Linear Ubiquitin Chain Assembly Complex MOLECULAR AND CELLULAR BIOLOGY Fujita, H., Rahighi, S., Akita, M., Kato, R., Sasaki, Y., Wakatsuki, S., Iwai, K. 2014; 34 (7): 1322-1335
Abstract

The linear ubiquitin chain assembly complex (LUBAC) ligase, consisting of HOIL-1L, HOIP, and SHARPIN, specifically generates linear polyubiquitin chains. LUBAC-mediated linear polyubiquitination has been implicated in NF-κB activation. NEMO, a component of the IκB kinase (IKK) complex, is a substrate of LUBAC, but the precise molecular mechanism underlying linear chain-mediated NF-κB activation has not been fully elucidated. Here, we demonstrate that linearly polyubiquitinated NEMO activates IKK more potently than unanchored linear chains. In mutational analyses based on the crystal structure of the complex between the HOIP NZF1 and NEMO CC2-LZ domains, which are involved in the HOIP-NEMO interaction, NEMO mutations that impaired linear ubiquitin recognition activity and prevented recognition by LUBAC synergistically suppressed signal-induced NF-κB activation. HOIP NZF1 bound to NEMO and ubiquitin simultaneously, and HOIP NZF1 mutants defective in interaction with either NEMO or ubiquitin could not restore signal-induced NF-κB activation. Furthermore, linear chain-mediated activation of IKK2 involved homotypic interaction of the IKK2 kinase domain. Collectively, these results demonstrate that linear polyubiquitination of NEMO plays crucial roles in IKK activation and that this modification involves the HOIP NZF1 domain and recognition of NEMO-conjugated linear ubiquitin chains by NEMO on another IKK complex.

View details for DOI 10.1128/MCB.01538-13

View details for Web of Science ID 000332696800013

View details for PubMedID 24469399
Expanded potential of seleno-carbohydrates as a molecular tool for X-ray structural determination of a carbohydrate-protein complex with single/multi-wavelength anomalous dispersion phasing. Bioorganic & medicinal chemistry Suzuki, T., Makyio, H., Ando, H., Komura, N., Menjo, M., Yamada, Y., Imamura, A., Ishida, H., Wakatsuki, S., Kato, R., Kiso, M. 2014; 22 (7): 2090-2101
Abstract

Seleno-lactoses have been successfully synthesized as candidates for mimicking carbohydrate ligands for human galectin-9 N-terminal carbohydrate recognition domain (NCRD). Selenium was introduced into the mono- or di-saccharides using p-methylselenobenzoic anhydride (Tol2Se) as a novel selenating reagent. The TolSe-substituted monosaccharides were converted into selenoglycosyl donors or acceptors, which were reacted with coupling partners to afford seleno-lactoses. The seleno-lactoses were converted to the target compounds. The structure of human galectin-9 NCRD co-crystallized with 6-MeSe-lactose was determined with single/multi-wavelength anomalous dispersion (SAD/MAD) phasing and was similar to that of the co-crystal with natural lactose.

View details for DOI 10.1016/j.bmc.2014.02.023

View details for PubMedID 24631362
Tuning Mechanism- Based Inactivators of Neuraminidases: Mechanistic and Structural Insights ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Buchini, S., Gallat, F., Greig, I. R., Kim, J., Wakatsuki, S., Chavas, L. M., Withers, S. G. 2014; 53 (13): 3382-3386
Abstract

3-Fluorosialosyl fluorides are inhibitors of sialidases that function by the formation of a long-lived covalent active-site adduct and have potential as therapeutics if made specific for the pathogen sialidase. Surprisingly, human Neu2 and the Trypanosoma cruzi trans-sialidase are inactivated more rapidly by the reagent with an equatorial fluorine at C3 than by its axial epimer, with reactivation following the same pattern. To explore a possible stereoelectronic basis for this, rate constants for spontaneous hydrolysis of the full series of four 3-fluorosialosyl fluorides were measured, and ground-state energies for each computed. The alpha (equatorial) anomeric fluorides hydrolyze more rapidly than their beta anomers, consistent with their higher ground-state energies. However ground-state energies do not explain the relative spontaneous reactivities of the 3-fluoro-epimers. The three-dimensional structures of the two 3-fluoro-sialosyl enzyme intermediates of human Neu2 were solved, revealing key stabilizing interactions between Arg21 and the equatorial, but not the axial, fluorine. Because of changes in geometry these interactions will increase at the transition state, likely explaining the difference in reaction rates.

View details for DOI 10.1002/anie.201309675

View details for Web of Science ID 000333001500014

View details for PubMedID 24591206
Structural Basis of the Autophagy-Related LC3/Atg13 LIR Complex: Recognition and Interaction Mechanism STRUCTURE Suzuki, H., Tabata, K., Morita, E., Kawasaki, M., Kato, R., Dobson, R. C., Yoshimori, T., Wakatsuki, S. 2014; 22 (1): 47-58
Abstract

Autophagy is a bulk degradation pathway that removes cytosolic materials to maintain cellular homeostasis. The autophagy-related gene 13 (Atg13) and microtubule associate protein 1 light chain 3 (LC3) proteins are required for autophagosome formation. We demonstrate that each of the human LC3 isoforms (LC3A, LC3B, and LC3C) interacts with Atg13 via the LC3 interacting region (LIR) of Atg13. Using X-ray crystallography, we solved the macromolecular structures of LC3A and LC3C, along with the complex structures of the LC3 isoforms with the Atg13 LIR. Together, our structural and binding analyses reveal that the side-chain of Lys49 of LC3 acts as a gatekeeper to regulate binding of the LIR. We verified this observation by mutation of Lys49 in LC3A, which significantly reduces LC3A positive puncta formation in cultured cells. Our results suggest that specific affinity of the LC3 isoforms to the Atg13 LIR is required for proper autophagosome formation.

View details for DOI 10.1016/j.str.2013.09.023

View details for Web of Science ID 000329593000007

View details for PubMedID 24290141
Improvement of an automated protein crystal exchange system PAM for high-throughput data collection JOURNAL OF SYNCHROTRON RADIATION Hiraki, M., Yamada, Y., Chavas, L. M., Wakatsuki, S., Matsugaki, N. 2013; 20: 890-893
Abstract

Photon Factory Automated Mounting system (PAM) protein crystal exchange systems are available at the following Photon Factory macromolecular beamlines: BL-1A, BL-5A, BL-17A, AR-NW12A and AR-NE3A. The beamline AR-NE3A has been constructed for high-throughput macromolecular crystallography and is dedicated to structure-based drug design. The PAM liquid-nitrogen Dewar can store a maximum of three SSRL cassettes. Therefore, users have to interrupt their experiments and replace the cassettes when using four or more of them during their beam time. As a result of investigation, four or more cassettes were used in AR-NE3A alone. For continuous automated data collection, the size of the liquid-nitrogen Dewar for the AR-NE3A PAM was increased, doubling the capacity. In order to check the calibration with the new Dewar and the cassette stand, calibration experiments were repeatedly performed. Compared with the current system, the parameters of the novel system are shown to be stable.

View details for DOI 10.1107/S0909049513021067

View details for Web of Science ID 000325639200016

View details for PubMedID 24121334
Improvements toward highly accurate diffraction experiments at the macromolecular micro-crystallography beamline BL-17A JOURNAL OF SYNCHROTRON RADIATION Yamada, Y., Chavas, L. M., Igarashi, N., Hiraki, M., Wakatsuki, S., Matsugaki, N. 2013; 20: 938-942
Abstract

BL-17A is a macromolecular crystallography beamline dedicated to diffraction experiments conducted using micro-crystals and structure determination studies using a lower energy X-ray beam. In these experiments, highly accurate diffraction intensity measurements are definitively important. Since this beamline was constructed, the beamline apparatus has been improved in several ways to enable the collection of accurate diffraction data. The stability of the beam intensities at the sample position was recently improved by modifying the monochromator. The diffractometer has also been improved. A new detector table was installed to prevent distortions in the diffractometer's base during the repositioning of the diffractometer detector. A new pinhole system and an on-axis viewing system were installed to improve the X-ray beam profile at the sample position and the centering of tiny crystal samples.

View details for DOI 10.1107/S0909049513022875

View details for Web of Science ID 000325639200026

View details for PubMedID 24121344
Structural basis for phosphorylation-triggered autophagic clearance of Salmonella. Biochemical journal Rogov, V. V., Suzuki, H., Fiskin, E., Wild, P., Kniss, A., Rozenknop, A., Kato, R., Kawasaki, M., McEwan, D. G., Löhr, F., Güntert, P., Dikic, I., Wakatsuki, S., Dötsch, V. 2013; 454 (3): 459-466
Abstract

Selective autophagy is mediated by the interaction of autophagy modifiers and autophagy receptors that also bind to ubiquitinated cargo. Optineurin is an autophagy receptor that plays a role in the clearance of cytosolic Salmonella. The interaction between receptors and modifiers is often relatively weak, with typical values for the dissociation constant in the low micromolar range. The interaction of optineurin with autophagy modifiers is even weaker, but can be significantly enhanced through phosphorylation by the TBK1 {TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)-associated nuclear factor κB activator]-binding kinase 1}. In the present study we describe the NMR and crystal structures of the autophagy modifier LC3B (microtubule-associated protein light chain 3 beta) in complex with the LC3 interaction region of optineurin either phosphorylated or bearing phospho-mimicking mutations. The structures show that the negative charge induced by phosphorylation is recognized by the side chains of Arg¹¹ and Lys⁵¹ in LC3B. Further mutational analysis suggests that the replacement of the canonical tryptophan residue side chain of autophagy receptors with the smaller phenylalanine side chain in optineurin significantly weakens its interaction with the autophagy modifier LC3B. Through phosphorylation of serine residues directly N-terminally located to the phenylalanine residue, the affinity is increased to the level normally seen for receptor-modifier interactions. Phosphorylation, therefore, acts as a switch for optineurin-based selective autophagy.

View details for PubMedID 23805866
Structural basis for phosphorylation-triggered autophagic clearance of Salmonella BIOCHEMICAL JOURNAL Rogov, V. V., Suzuki, H., Fiskin, E., Wild, P., Kniss, A., Rozenknop, A., Kato, R., Kawasaki, M., McEwan, D. G., Loehr, F., Guentert, P., Dikic, I., Wakatsuki, S., Doetsch, V. 2013; 454: 459-466
View details for DOI 10.1042/BJ20121907

View details for Web of Science ID 000329975400010
Structures of an ATP-independent Lon-like protease and its complexes with covalent inhibitors ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Liao, J., Ihara, K., Kuo, C., Huang, K., Wakatsuki, S., Wu, S., Chang, C. 2013; 69: 1395-1402
Abstract

The Lon proteases are a unique family of chambered proteases with a built-in AAA+ (ATPases associated with diverse cellular activities) module. Here, crystal structures of a unique member of the Lon family with no intrinsic ATPase activity in the proteolytically active form are reported both alone and in complexes with three covalent inhibitors: two peptidomimetics and one derived from a natural product. This work reveals the unique architectural features of an ATP-independent Lon that selectively degrades unfolded protein substrates. Importantly, these results provide mechanistic insights into the recognition of inhibitors and polypeptide substrates within the conserved proteolytic chamber, which may aid the development of specific Lon-protease inhibitors.

View details for DOI 10.1107/S0907444913008214

View details for Web of Science ID 000322445100007

View details for PubMedID 23897463
Structural basis of preferential binding of fucose-containing saccharide by the Caenorhabditis elegans galectin LEC-6 GLYCOBIOLOGY Makyio, H., Takeuchi, T., Tamura, M., Nishiyama, K., Takahashi, H., Natsugari, H., Arata, Y., Kasai, K., Yamada, Y., Wakatsuki, S., Kato, R. 2013; 23 (7): 797-805
Abstract

Galectins are a group of lectins that can bind carbohydrate chains containing β-galactoside units. LEC-6, a member of galectins of Caenorhabditis elegans, binds fucose-containing saccharides. We solved the crystal structure of LEC-6 in complex with galactose-β1,4-fucose (Galβ1-4Fuc) at 1.5 Å resolution. The overall structure of the protein and the identities of the amino-acid residues binding to the disaccharide are similar to those of other galectins. However, further structural analysis and multiple sequence alignment between LEC-6 and other galectins indicate that a glutamic acid residue (Glu67) is important for the preferential binding between LEC-6 and the fucose moiety of the Galβ1-4Fuc unit. Frontal affinity chromatography analysis indicated that the affinities of E67D and E67A mutants for Galβ1-4Fuc are lower than that of wild-type LEC-6. Furthermore, the affinities of Glu67 mutants for an endogenous oligosaccharide, which contains a Galβ1-4Fuc unit, are drastically reduced relative to that of the wild-type protein. We conclude that the Glu67 in the oligosaccharide-binding site assists the recognition of the fucose moiety by LEC-6.

View details for DOI 10.1093/glycob/cwt017

View details for Web of Science ID 000320124400004

View details for PubMedID 23481096
Direct metal recognition by guanine nucleotide-exchange factor in the initial step of the exchange reaction ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Uejima, T., Ihara, K., Sunada, M., Kawasaki, M., Ueda, T., Kato, R., Nakano, A., Wakatsuki, S. 2013; 69: 345-351
Abstract

Rab small GTPases regulate vesicle transport in eukaryotes by interacting with various effectors. Guanine nucleotide-exchange factor (GEF) catalyzes the transition from inactive GDP-bound Rab to active GTP-bound Rab. The existence of several GDP-bound intermediates containing the Arabidopsis thaliana Rab5 homologue ARA7 and the GEF VPS9a prior to the formation of a nucleotide-free binary complex has been proposed [Uejima et al. (2010), J. Biol. Chem. 285, 36689-36697]. During this process, VPS9a directly interacts with the β-phosphate of GDP and the P-loop lysine of ARA7 via a catalytically important aspartate finger, which promotes the release of GDP from ARA7. However, it is unclear how VPS9a removes Mg2+ from ARA7 before forming the GDP-bound ternary complex. Here, the structure of the ARA7-GDP-Ca2+-VPS9a complex is reported, in which the aspartate finger directly coordinates the divalent metal ion. Ca2+ is bound to the canonical Mg2+-binding site, coordinated by the β-phosphate of GDP and the P-loop serine of ARA7. Unexpectedly, Ca2+ is further coordinated by the aspartate finger and the main chain of VPS9a. This structure may represent the earliest intermediate step in the GEF-catalyzed nucleotide-exchange reaction of ARA7 before the metal-free GDP-bound intermediates are created.

View details for DOI 10.1107/S0907444912047294

View details for Web of Science ID 000316742700004

View details for PubMedID 23519409
Structural switching of Cu,Zn-superoxide dismutases at loop VI: insights from the crystal structure of 2-mercaptoethanol-modified enzyme BIOSCIENCE REPORTS Ihara, K., Fujiwara, N., Yamaguchi, Y., Torigoe, H., Wakatsuki, S., Taniguchi, N., Suzuki, K. 2012; 32 (6): 539-548
Abstract

Cu,Zn SOD1 (superoxide dismutase 1) is implicated in FALS (familial amyotrophic lateral sclerosis) through the accumulation of misfolded proteins that are toxic to neuronal cells. Loop VI (residues 102-115) of the protein is at the dimer interface and could play a critical role in stability. The free cysteine residue, Cys111 in the loop, is readily oxidized and alkylated. We have found that modification of this Cys111 with 2-ME (2-mercaptoethanol; 2-ME-SOD1) stabilizes the protein and the mechanism may provide insights into destabilization and the formation of aggregated proteins. Here, we determined the crystal structure of 2-ME-SOD1 and find that the 2-ME moieties in both subunits interact asymmetrically at the dimer interface and that there is an asymmetric configuration of segment Gly108 to Cys111 in loop VI. One loop VI of the dimer forms a 310-helix (Gly108 to His110) within a unique β-bridge stabilized by a hydrogen bond between Ser105-NH and His110-CO, while the other forms a β-turn without the H-bond. The H-bond (H-type) and H-bond free (F-type) configurations are also seen in some wild-type and mutant human SOD1s in the Protein Data Bank suggesting that they are interconvertible and an intrinsic property of SOD1s. The two structures serve as a basis for classification of these proteins and hopefully a guide to their stability and role in pathophysiology.

View details for DOI 10.1042/BSR20120029

View details for Web of Science ID 000312281400004

View details for PubMedID 22804629
Crystal sample pins and a storage cassette system compatible with the protein crystallography beamlines at both the Photon Factory and SPring-8 JOURNAL OF APPLIED CRYSTALLOGRAPHY Fujihashi, M., Hiraki, M., Ueno, G., Baba, S., Murakami, H., Suzuki, M., Watanabe, N., Tanaka, I., Nakagawa, A., Wakatsuki, S., Yamamoto, M., Miki, K. 2012; 45: 1156-1161
View details for DOI 10.1107/S002188981203734X

View details for Web of Science ID 000311293100008
International Workshop on Improving Data Quality and Quantity for XAFS Experiments (Q2XAFS 2011) JOURNAL OF SYNCHROTRON RADIATION Ascone, I., Asakura, K., George, G. N., Wakatsuki, S. 2012; 19: 849-850
View details for DOI 10.1107/S0909049512043506

View details for Web of Science ID 000310151000001

View details for PubMedID 23093741
Frontiers and challenges of biophysical methods: from computational biology to X-ray free electron laser. Current opinion in structural biology Hasnain, S. S., Wakatsuki, S. 2012; 22 (5): 591-593
View details for DOI 10.1016/j.sbi.2012.09.005

View details for PubMedID 23036819
Structural Basis for Membrane Binding Specificity of the Bin/Amphiphysin/Rvs (BAR) Domain of Arfaptin-2 Determined by Arl1 GTPase JOURNAL OF BIOLOGICAL CHEMISTRY Nakamura, K., Man, Z., Xie, Y., Hanai, A., Makyio, H., Kawasaki, M., Kato, R., Shin, H., Nakayama, K., Wakatsuki, S. 2012; 287 (30): 25478-25489
Abstract

Membrane-sculpting BAR (Bin/Amphiphysin/Rvs) domains form a crescent-shaped homodimer that can sense and induce membrane curvature through its positively charged concave face. We have recently shown that Arfaptin-2, which was originally identified as a binding partner for the Arf and Rac1 GTPases, binds to Arl1 through its BAR domain and is recruited onto Golgi membranes. There, Arfaptin-2 induces membrane tubules. Here, we report the crystal structure of the Arfaptin-2 BAR homodimer in complex with two Arl1 molecules bound symmetrically to each side, leaving the concave face open for membrane association. The overall structure of the Arl1·Arfaptin-2 BAR complex closely resembles that of the PX-BAR domain of sorting nexin 9, suggesting similar mechanisms underlying BAR domain targeting to specific organellar membranes. The Arl1·Arfaptin-2 BAR structure suggests that one of the two Arl1 molecules competes with Rac1, which binds to the concave face of the Arfaptin-2 BAR homodimer and may hinder its membrane association.

View details for DOI 10.1074/jbc.M112.365783

View details for Web of Science ID 000306651700054

View details for PubMedID 22679020
Structural basis for Arf6-MKLP1 complex formation on the Flemming body responsible for cytokinesis EMBO JOURNAL Makyio, H., Ohgi, M., Takei, T., Takahashi, S., Takatsu, H., Katoh, Y., Hanai, A., Ueda, T., Kanaho, Y., Xie, Y., Shin, H., Kamikubo, H., Kataoka, M., Kawasaki, M., Kato, R., Wakatsuki, S., Nakayama, K. 2012; 31 (11): 2590-2603
Abstract

A small GTPase, Arf6, is involved in cytokinesis by localizing to the Flemming body (the midbody). However, it remains unknown how Arf6 contributes to cytokinesis. Here, we demonstrate that Arf6 directly interacts with mitotic kinesin-like protein 1 (MKLP1), a Flemming body-localizing protein essential for cytokinesis. The crystal structure of the Arf6-MKLP1 complex reveals that MKLP1 forms a homodimer flanked by two Arf6 molecules, forming a 2:2 heterotetramer containing an extended β-sheet composed of 22 β-strands that spans the entire heterotetramer, suitable for interaction with a concave membrane surface at the cleavage furrow. We show that, during cytokinesis, Arf6 is first accumulated around the cleavage furrow and, prior to abscission, recruited onto the Flemming body via interaction with MKLP1. We also show by structure-based mutagenesis and siRNA-mediated knockdowns that the complex formation is required for completion of cytokinesis. A model based on these results suggests that the Arf6-MKLP1 complex plays a crucial role in cytokinesis by connecting the microtubule bundle and membranes at the cleavage plane.

View details for DOI 10.1038/emboj.2012.89

View details for Web of Science ID 000304600300016

View details for PubMedID 22522702
S-SAD phasing study of death receptor 6 and its solution conformation revealed by SAXS ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Ru, H., Zhao, L., Ding, W., Jiao, L., Shaw, N., Liang, W., Zhang, L., Hung, L., Matsugaki, N., Wakatsuki, S., Liu, Z. 2012; 68: 521-530
Abstract

A subset of tumour necrosis factor receptor (TNFR) superfamily members contain death domains in their cytoplasmic tails. Death receptor 6 (DR6) is one such member and can trigger apoptosis upon the binding of a ligand by its cysteine-rich domains (CRDs). The crystal structure of the ectodomain (amino acids 1-348) of human death receptor 6 (DR6) encompassing the CRD region was phased using the anomalous signal from S atoms. In order to explore the feasibility of S-SAD phasing at longer wavelengths (beyond 2.5 Å), a comparative study was performed on data collected at wavelengths of 2.0 and 2.7 Å. In spite of sub-optimal experimental conditions, the 2.7 Å wavelength used for data collection showed potential for S-SAD phasing. The results showed that the R(ano)/R(p.i.m.) ratio is a good indicator for monitoring the anomalous data quality when the anomalous signal is relatively strong, while d''/sig(d'') calculated by SHELXC is a more sensitive and stable indicator applicable for grading a wider range of anomalous data qualities. The use of the `parameter-space screening method' for S-SAD phasing resulted in solutions for data sets that failed during manual attempts. SAXS measurements on the ectodomain suggested that a dimer defines the minimal physical unit of an unliganded DR6 molecule in solution.

View details for DOI 10.1107/S0907444912004490

View details for Web of Science ID 000303159000004

View details for PubMedID 22525750
Beamline AR-NW12A: high-throughput beamline for macromolecular crystallography at the Photon Factory JOURNAL OF SYNCHROTRON RADIATION Chavas, L. M., Matsugaki, N., Yamada, Y., Hiraki, M., Igarashi, N., Suzuki, M., Wakatsuki, S. 2012; 19: 450-454
Abstract

AR-NW12A is an in-vacuum undulator beamline optimized for high-throughput macromolecular crystallography experiments as one of the five macromolecular crystallography (MX) beamlines at the Photon Factory. This report provides details of the beamline design, covering its optical specifications, hardware set-up, control software, and the latest developments for MX experiments. The experimental environment presents state-of-the-art instrumentation for high-throughput projects with a high-precision goniometer with an adaptable goniometer head, and a UV-light sample visualization system. Combined with an efficient automounting robot modified from the SSRL SAM system, a remote control system enables fully automated and remote-access X-ray diffraction experiments.

View details for DOI 10.1107/S0909049512009727

View details for Web of Science ID 000302998900023

View details for PubMedID 22514184
Structural basis of the strict phospholipid binding specificity of the pleckstrin homology domain of human evectin-2 ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Okazaki, S., Kato, R., Uchida, Y., Taguchi, T., Arai, H., Wakatsuki, S. 2012; 68: 117-123
Abstract

Evectin-2 is a recycling endosomal protein involved in retrograde transport. Its primary sequence contains an N-terminal pleckstrin homology (PH) domain and a C-terminal hydrophobic region. The PH domain of evectin-2 can specifically bind phosphatidylserine, which is enriched in recycling endosomes, and plays an essential role in retrograde transport from recycling endosomes to the trans-Golgi network. The structure of human evectin-2 PH domain in complex with O-phospho-L-serine has recently been reported and demonstrates how the head group of phosphatidylserine is recognized. However, it was not possible to elucidate from the structure why evectin-2 cannot bind phosphatidic acid or phosphatidylethanolamine, which share a common moiety with phosphatidylserine. Here, the crystal structure at 1.75 Å resolution of an apo form of human evectin-2 PH domain, in which the ligand-binding site is free from crystal packing and is thus appropriate for comparison with the structure of the complex, is reported. Comparison between the structures of the apo form and the O-phospho-L-serine complex revealed ligand-induced conformational change evoked by interaction between the carboxyl moiety of the head group of phosphatidylserine and the main-chain N atom of Thr14. This structural change effectively explains the strict ligand specificity of the PH domain of human evectin-2.

View details for DOI 10.1107/S0907444911051626

View details for Web of Science ID 000299469100004

View details for PubMedID 22281740
Structure of a compact conformation of linear diubiquitin ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Rohaim, A., Kawasaki, M., Kato, R., Dikic, I., Wakatsuki, S. 2012; 68: 102-108
Abstract

Post-translational modifications involving ubiquitin regulate a wide range of biological processes including protein degradation, responses to DNA damage and immune signalling. Ubiquitin polymerizes into chains which may contain eight different linkage types; the ubiquitin C-terminal glycine can link to one of the seven lysine residues or the N-terminal amino group of methionine in the distal ubiquitin molecule. The latter head-to-tail linkage type, referred to as a linear ubiquitin chain, is involved in NF-κB activation through specific interactions with NF-κB essential modulator (NEMO). Here, a crystal structure of linear diubiquitin at a resolution of 2.2 Å is reported. Although the two ubiquitin moieties do not interact with each other directly, the overall structure adopts a compact but not completely closed conformation with a few intermoiety contacts. This structure differs from the previously reported extended conformation, which resembles Lys63-linked diubiquitin, suggesting that the linear polyubiquitin chain is intrinsically flexible and can adopt multiple conformations.

View details for DOI 10.1107/S0907444911051195

View details for Web of Science ID 000299469100002

View details for PubMedID 22281738
Intracellular phosphatidylserine is essential for retrograde membrane traffic through endosomes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Uchida, Y., Hasegawa, J., Chinnapen, D., Inoue, T., Okazaki, S., Kato, R., Wakatsuki, S., Misaki, R., Koike, M., Uchiyama, Y., Iemura, S., Natsume, T., Kuwahara, R., Nakagawa, T., Nishikawa, K., Mukai, K., Miyoshi, E., Taniguchi, N., Sheff, D., Lencer, W. I., Taguchi, T., Arai, H. 2011; 108 (38): 15846-15851
Abstract

Phosphatidylserine (PS) is a relatively minor constituent of biological membranes. Despite its low abundance, PS in the plasma membrane (PM) plays key roles in various phenomena such as the coagulation cascade, clearance of apoptotic cells, and recruitment of signaling molecules. PS also localizes in endocytic organelles, but how this relates to its cellular functions remains unknown. Here we report that PS is essential for retrograde membrane traffic at recycling endosomes (REs). PS was most concentrated in REs among intracellular organelles, and evectin-2 (evt-2), a protein of previously unknown function, was targeted to REs by the binding of its pleckstrin homology (PH) domain to PS. X-ray analysis supported the specificity of the binding of PS to the PH domain. Depletion of evt-2 or masking of intracellular PS suppressed membrane traffic from REs to the Golgi. These findings uncover the molecular basis that controls the RE-to-Golgi transport and identify a unique PH domain that specifically recognizes PS but not polyphosphoinositides.

View details for DOI 10.1073/pnas.1109101108

View details for Web of Science ID 000295030000040

View details for PubMedID 21911378
Improved Inactivation Effect of Bacteria: Fabrication of Mesoporous Anatase Films with Fine Ag Nanoparticles Prepared by Coaxial Vacuum Arc Deposition CHEMISTRY LETTERS Oveisi, H., Rahighi, S., Jiang, X., Agawa, Y., Beitollahi, A., Wakatsuki, S., Yamauchi, Y. 2011; 40 (4): 420-422
View details for DOI 10.1246/cl.2011.420

View details for Web of Science ID 000290552200032
UV LED lighting for automated crystal centring JOURNAL OF SYNCHROTRON RADIATION Chavas, L. M., Yamada, Y., Hiraki, M., Igarashi, N., Matsugaki, N., Wakatsuki, S. 2011; 18: 11-15
Abstract

A direct outcome of the exponential growth of macromolecular crystallography is the continuously increasing demand for synchrotron beam time, both from academic and industrial users. As more and more projects entail screening a profusion of sample crystals, fully automated procedures at every level of the experiments are being implemented at all synchrotron facilities. One of the major obstacles to achieving such automation lies in the sample recognition and centring in the X-ray beam. The capacity of UV light to specifically react with aromatic residues present in proteins or with DNA base pairs is at the basis of UV-assisted crystal centring. Although very efficient, a well known side effect of illuminating biological samples with strong UV sources is the damage induced on the irradiated samples. In the present study the effectiveness of a softer UV light for crystal centring by taking advantage of low-power light-emitting diode (LED) sources has been investigated. The use of UV LEDs represents a low-cost solution for crystal centring with high specificity.

View details for DOI 10.1107/S0909049510028670

View details for Web of Science ID 000285409000004

View details for PubMedID 21169682
Selective Binding of Linear Ubiquitin Chains to NEMO in NF-kappaB Activation 12th Biennial International Tumor Necrosis Factor Conference Ikeda, F., Rahighi, S., Wakatsuki, S., Dikic, I. SPRINGER-VERLAG BERLIN. 2011: 107–114
View details for DOI 10.1007/978-1-4419-6612-4_11

View details for Web of Science ID 000291501300013

View details for PubMedID 21153314
GDP-bound and Nucleotide-free Intermediates of the Guanine Nucleotide Exchange in the Rab5.Vps9 System JOURNAL OF BIOLOGICAL CHEMISTRY Uejima, T., Ihara, K., Goh, T., Ito, E., Sunada, M., Ueda, T., Nakano, A., Wakatsuki, S. 2010; 285 (47): 36689-36697
Abstract

Many GTPases regulate intracellular transport and signaling in eukaryotes. Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing the exchange of their GDP for GTP. Here we present crystallographic and biochemical studies of a GEF reaction with four crystal structures of Arabidopsis thaliana ARA7, a plant homolog of Rab5 GTPase, in complex with its GEF, VPS9a, in the nucleotide-free and GDP-bound forms, as well as a complex with aminophosphonic acid-guanylate ester and ARA7·VPS9a(D185N) with GDP. Upon complex formation with ARA7, VPS9 wedges into the interswitch region of ARA7, inhibiting the coordination of Mg(2+) and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP β-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP β-phosphate toward switch II to further destabilize GDP for its release during the transition from the GDP-bound to nucleotide-free intermediates in the nucleotide exchange reaction.

View details for DOI 10.1074/jbc.M110.152132

View details for Web of Science ID 000284146100070

View details for PubMedID 20833725
Advances in biophysical methods: characterisation and visualization of molecules, cells and organism CURRENT OPINION IN STRUCTURAL BIOLOGY Hasnain, S. S., Wakatsuki, S. 2010; 20 (5): 584-586
View details for DOI 10.1016/j.sbi.2010.09.004

View details for Web of Science ID 000284676900008

View details for PubMedID 20884198
Crystallization of small proteins assisted by green fluorescent protein ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Suzuki, N., Hiraki, M., Yamada, Y., Matsugaki, N., Igarashi, N., Kato, R., Dikic, I., Drew, D., Iwata, S., Wakatsuki, S., Kawasaki, M. 2010; 66: 1059-1066
Abstract

The generation of crystal lattice contacts by proteinaceous tags fused to target proteins is an attractive approach to aid in the crystallization of otherwise intractable proteins. Here, the use of green fluorescent protein (GFP) fusions for this purpose is demonstrated, using ubiquitin and the ubiquitin-binding motif (UBM) of Y-family polymerase ι as examples. The structure of the GFP-ubiquitin fusion protein revealed that the crystal lattice was formed by GFP moieties. Ubiquitin was accommodated in the lattice through interactions with the peripheral loops of GFP. However, in the GFP-UBM fusion crystal UBM formed extensive interactions with GFP and these interactions, together with UBM dimerization, mediated the crystal packing. Interestingly, the tyrosine residues that are involved in mediating crystal contacts in both GFP-ubiquitin and GFP-UBM crystals are arranged in a belt on the surface of the β-barrel structure of GFP. Therefore, it is likely that GFP can assist in the crystallization of small proteins and of protein domains in general.

View details for DOI 10.1107/S0907444910032944

View details for Web of Science ID 000283167400003

View details for PubMedID 20944239
Crystal structure of cyclic Lys48-linked tetraubiquitin BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Satoh, T., Sakata, E., Yamamoto, S., Yamaguchi, Y., Sumiyoshi, A., Wakatsuki, S., Kato, K. 2010; 400 (3): 329-333
Abstract

Lys48-linked polyubiquitin chains serve as a signal for protein degradation by 26S proteasomes through its Ile44 hydrophobic patches interactions. The individual ubiquitin units of each chain are conjugated through an isopeptide bond between Lys48 and the C-terminal Gly76 of the preceding units. The conformation of Lys48-linked tetraubiquitin has been shown to change dynamically depending on solution pH. Here we enzymatically synthesized a wild-type Lys48-linked tetraubiquitin for structural study. In the synthesis, cyclic and non-cyclic species were obtained as major and minor fractions, respectively. This enabled us to solve the crystal structure of tetraubiquitin exclusively with native Lys48-linkages at 1.85A resolution in low pH 4.6. The crystallographic data clearly showed that the C-terminus of the first ubiquitin is conjugated to the Lys48 residue of the fourth ubiquitin. The overall structure is quite similar to the closed form of engineered tetraubiquitin at near-neutral pH 6.7, previously reported, in which the Ile44 hydrophobic patches face each other. The structure of the second and the third ubiquitin units [Ub(2)-Ub(3)] connected through a native isopeptide bond is significantly different from the conformations of the corresponding linkage of the engineered tetraubiquitins, whereas the structures of Ub(1)-Ub(2) and Ub(3)-Ub(4) isopeptide bonds are almost identical to those of the previously reported structures. From these observations, we suggest that the flexible nature of the isopeptide linkage thus observed contributes to the structural arrangements of ubiquitin chains exemplified by the pH-dependent closed-to-open conformational transition of tetraubiquitin.

View details for DOI 10.1016/j.bbrc.2010.08.057

View details for Web of Science ID 000282616200007

View details for PubMedID 20728431
Unusual Antibacterial Property of Mesoporous Titania Films: Drastic Improvement by Controlling Surface Area and Crystallinity CHEMISTRY-AN ASIAN JOURNAL Oveisi, H., Rahighi, S., Jiang, X., Nemoto, Y., Beitollahi, A., Wakatsuki, S., Yamauchi, Y. 2010; 5 (9): 1978-1983
View details for DOI 10.1002/asia.201000351

View details for Web of Science ID 000281916600006

View details for PubMedID 20665652
Molecular basis for defect in Alix-binding by alternatively spliced isoform of ALG-2 (ALG-2(Delta GF122)) and structural roles of F122 in target recognition BMC STRUCTURAL BIOLOGY Inuzuka, T., Suzuki, H., Kawasaki, M., Shibata, H., Wakatsuki, S., Maki, M. 2010; 10
Abstract

ALG-2 (a gene product of PDCD6) belongs to the penta-EF-hand (PEF) protein family and Ca2+-dependently interacts with various intracellular proteins including mammalian Alix, an adaptor protein in the ESCRT system. Our previous X-ray crystal structural analyses revealed that binding of Ca2+ to EF3 enables the side chain of R125 to move enough to make a primary hydrophobic pocket (Pocket 1) accessible to a short fragment of Alix. The side chain of F122, facing a secondary hydrophobic pocket (Pocket 2), interacts with the Alix peptide. An alternatively spliced shorter isoform, designated ALG-2DeltaGF122, lacks Gly121Phe122 and does not bind Alix, but the structural basis of the incompetence has remained to be elucidated.We solved the X-ray crystal structure of the PEF domain of ALG-2DeltaGF122 in the Ca2+-bound form and compared it with that of ALG-2. Deletion of the two residues shortened alpha-helix 5 (alpha5) and changed the configuration of the R125 side chain so that it partially blocked Pocket 1. A wall created by the main chain of 121-GFG-123 and facing the two pockets was destroyed. Surprisingly, however, substitution of F122 with Ala or Gly, but not with Trp, increased the Alix-binding capacity in binding assays. The F122 substitutions exhibited different effects on binding of ALG-2 to other known interacting proteins, including TSG101 (Tumor susceptibility gene 101) and annexin A11. The X-ray crystal structure of the F122A mutant revealed that removal of the bulky F122 side chain not only created an additional open space in Pocket 2 but also abolished inter-helix interactions with W95 and V98 (present in alpha4) and that alpha5 inclined away from alpha4 to expand Pocket 2, suggesting acquirement of more appropriate positioning of the interacting residues to accept Alix.We found that the inability of the two-residue shorter ALG-2 isoform to bind Alix is not due to the absence of bulky side chain of F122 but due to deformation of a main-chain wall facing pockets 1 and 2. Moreover, a residue at the position of F122 contributes to target specificity and a smaller side chain is preferable for Alix binding but not favored to bind annexin A11.

View details for DOI 10.1186/1472-6807-10-25

View details for Web of Science ID 000282254900001

View details for PubMedID 20691033
Advancement of Synchrotron Radiation Protein Crystallography Aimed by the Targeted Protein Research Program: Beamline Developments at the Photon Factory YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN Wakatsuki, S., Yamada, Y., Chavas, L. M., Igarashi, N., Kawasaki, M., Kato, R., Hiraki, M., Matsugaki, N. 2010; 130 (5): 631-640
Abstract

The Targeted Protein Research Program (TPRP) started in 2007 as a sequel of the Protein 3000 Project which lasted from 2002 to 2007. In the new project, four cores, Protein Production, Structure Analysis, Control of Protein Functions with Compounds, and Informatics, have been established as focus of methodology developments critical for functional and structural studies by the target protein research teams. Within the "Analysis Core" synchrotron radiation plays a pivotal role providing X-ray beams for structural analyses of the target proteins. The two large Japanese synchrotron radiation facilities, SPring-8 and Photon Factory (PF), along with three protein crystallography groups from Hokkaido, Kyoto and Osaka Universities have teamed up to develop two complementary micro-beam beamlines, one on each synchrotron site, and associated technologies for cutting edge structural biology research. At the PF, there are 5 operational beamlines which are equipped with state-of-the-art instrumentation for high-throughput protein crystallography experiments. Within the TPRP framework, the PF is developing a micro-focus beamline optimized for a lower energy single anomalous diffraction (SAD) experiment. This will be particularly useful for structure determination of difficult protein targets for which heavy atom derivatives or selenomethionine substitution does not work and other standard phasing methods fail to give structure solutions. This will augment the capabilities of the PF structural biology beamlines with similar look-and-feel experimental environments.

View details for Web of Science ID 000277008000002

View details for PubMedID 20460857
Complexity in Influenza Virus Targeted Drug Design: Interaction with Human Sialidases JOURNAL OF MEDICINAL CHEMISTRY Chavas, L. M., Kato, R., Suzuki, N., von Itzstein, M., Mann, M. C., Thomson, R. J., Dyason, J. C., McKimm-Breschkin, J., Fusi, P., Tringali, C., Venerando, B., Tettamanti, G., Monti, E., Wakatsuki, S. 2010; 53 (7): 2998-3002
Abstract

With the global spread of the pandemic H1N1 and the ongoing pandemic potential of the H5N1 subtype, the influenza virus represents one of the most alarming viruses spreading worldwide. The influenza virus sialidase is an effective drug target, and a number of inhibitors are clinically effective against the virus (zanamivir, oseltamivir, peramivir). Here we report structural and biochemical studies of the human cytosolic sialidase Neu2 with influenza virus sialidase-targeting drugs and related compounds.

View details for DOI 10.1021/jm100078r

View details for Web of Science ID 000276096300027

View details for PubMedID 20222714
Structural basis for the cooperative interplay between the two causative gene products of combined factor V and factor VIII deficiency PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Nishio, M., Kamiya, Y., Mizushima, T., Wakatsuki, S., Sasakawa, H., Yamamoto, K., Uchiyama, S., Noda, M., Mckay, A. R., Fukui, K., Hauri, H., Kato, K. 2010; 107 (9): 4034-4039
Abstract

Combined deficiency of coagulation factors V and VIII (F5F8D), an autosomal recessive disorder characterized by coordinate reduction in the plasma levels of factor V (FV) and factor VIII (FVIII), is genetically linked to mutations in the transmembrane lectin ERGIC-53 and the soluble calcium-binding protein MCFD2. Growing evidence indicates that these two proteins form a complex recycling between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment and thereby function as a cargo receptor in the early secretory pathway of FV and FVIII. For better understanding of the mechanisms underlying the functional coordination of ERGIC-53 and MCFD2, we herein characterize their interaction by x-ray crystallographic analysis in conjunction with NMR and ultracentrifugation analyses. Inspection of the combined data reveals that ERGIC-53-CRD binds MCFD2 through its molecular surface remote from the sugar-binding site, giving rise to a 11 complex in solution. The interaction is independent of sugar-binding of ERGIC-53 and involves most of the missense mutation sites of MCFD2 so far reported in F5F8D. Comparison with the previously reported uncomplexed structure of each protein indicates that MCFD2 but not ERGIC-53-CRD undergoes significant conformational alterations upon complex formation. Our findings provide a structural basis for the cooperative interplay between ERGIC-53 and MCFD2 in capturing FV and FVIII.

View details for DOI 10.1073/pnas.0908526107

View details for Web of Science ID 000275131100019

View details for PubMedID 20142513
Crystal Structures of the CERT START Domain with Inhibitors Provide Insights into the Mechanism of Ceramide Transfer JOURNAL OF MOLECULAR BIOLOGY Kudo, N., Kumagai, K., Matsubara, R., Kobayashi, S., Hanada, K., Wakatsuki, S., Kato, R. 2010; 396 (2): 245-251
Abstract

The cytosolic protein CERT transfers ceramide from the endoplasmic reticulum to the Golgi apparatus where ceramide is converted to SM. The C-terminal START (steroidogenic acute regulatory protein-related lipid transfer) domain of CERT binds one ceramide molecule in its central amphiphilic cavity. (1R,3R)-N-(3-Hydroxy-1-hydroxymethyl-3-phenylpropyl)alkanamide (HPA), a synthesized analogue of ceramide, inhibits ceramide transfer by CERT. Here we report crystal structures of the CERT START domain in complex with HPAs of varying acyl chain lengths. In these structures, one HPA molecule is buried in the amphiphilic cavity where the amide and hydroxyl groups of HPA form a hydrogen-bond network with specific amino acid residues. The Omega1 loop, which has been suggested to function as a gate of the cavity, adopts a different conformation when bound to HPA than when bound to ceramide. In the Omega1 loop region, Trp473 shows the largest difference between these two structures. This residue exists inside of the cavity in HPA-bound structures, while it is exposed to the outside of the protein in the apo-form and ceramide-bound complex structures. Surface plasmon resonance experiments confirmed that Trp473 is important for interaction with membranes. These results provide insights into not only the molecular mechanism of inhibition by HPAs but also possible mechanisms by which CERT interacts with ceramide.

View details for DOI 10.1016/j.jmb.2009.12.029

View details for Web of Science ID 000274980400001

View details for PubMedID 20036255
Crystal Structure of UbcH5b similar to Ubiquitin Intermediate: Insight into the Formation of the Self-Assembled E2 similar to Ub Conjugates STRUCTURE Sakata, E., Satoh, T., Yamamoto, S., Yamaguchi, Y., Yagi-Utsumi, M., Kurimoto, E., Tanaka, K., Wakatsuki, S., Kato, K. 2010; 18 (1): 138-147
Abstract

E2 ubiquitin-conjugating enzymes catalyze the attachment of ubiquitin to lysine residues of target proteins. The UbcH5b E2 enzyme has been shown to play a key role in the initiation of the ubiquitination of substrate proteins upon action of several E3 ligases. Here we have determined the 2.2 A crystal structure of an intermediate of UbcH5b~ubiquitin (Ub) conjugate, which is assembled into an infinite spiral through the backside interaction. This active complex may provide multiple E2 active sites, enabling efficient ubiquitination of substrates. Indeed, biochemical assays support a model in which the self-assembled UbcH5b~Ub can serve as a bridge for the gap between the lysine residue of the substrate and the catalytic cysteine of E2.

View details for DOI 10.1016/j.str.2009.11.007

View details for Web of Science ID 000273859700017

View details for PubMedID 20152160
Ubiquitin-binding domains - from structures to functions NATURE REVIEWS MOLECULAR CELL BIOLOGY Dikic, I., Wakatsuki, S., Walters, K. J. 2009; 10 (10): 659-671
Abstract

Ubiquitin-binding domains (UBDs) are modular elements that bind non-covalently to the protein modifier ubiquitin. Recent atomic-level resolution structures of ubiquitin-UBD complexes have revealed some of the mechanisms that underlie the versatile functions of ubiquitin in vivo. The preferences of UBDs for ubiquitin chains of specific length and linkage are central to these functions. These preferences originate from multimeric interactions, whereby UBDs synergistically bind multiple ubiquitin molecules, and from contacts with regions that link ubiquitin molecules into a polymer. The sequence context of UBDs and the conformational changes that follow their binding to ubiquitin also contribute to ubiquitin signalling. These new structure-based insights provide strategies for controlling cellular processes by targeting ubiquitin-UBD interfaces.

View details for DOI 10.1038/nrm2767

View details for Web of Science ID 000270040300009

View details for PubMedID 19773779
[Structure determination of proteins without preparing derivative crystals]. Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme Matsugaki, N., Yamada, Y., Hiraki, M., Igarashi, N., Wakatsuki, S. 2009; 54 (12): 1484-1489
View details for PubMedID 21089575
[Development of beam line X-ray crystallography targeting multiprotein complexes: Overview]. Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme Wakatsuki, S., Yamamoto, M., Tanaka, I., Miki, K., Nakagawa, A. 2009; 54 (12): 1476-?
View details for PubMedID 21089573
Specific Recognition of Linear Ubiquitin Chains by NEMO Is Important for NF-kappa B Activation CELL Rahighi, S., Ikeda, F., Kawasaki, M., Akutsu, M., Suzuki, N., Kato, R., Kensche, T., Uejima, T., Bloor, S., Komander, D., Randow, F., Wakatsuki, S., Dikic, I. 2009; 136 (6): 1098-1109
Abstract

Activation of nuclear factor-kappaB (NF-kappaB), a key mediator of inducible transcription in immunity, requires binding of NF-kappaB essential modulator (NEMO) to ubiquitinated substrates. Here, we report that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO selectively binds linear (head-to-tail) ubiquitin chains. Crystal structures of the UBAN motif revealed a parallel coiled-coil dimer that formed a heterotetrameric complex with two linear diubiquitin molecules. The UBAN dimer contacted all four ubiquitin moieties, and the integrity of each binding site was required for efficient NF-kappaB activation. Binding occurred via a surface on the proximal ubiquitin moiety and the canonical Ile44 surface on the distal one, thereby providing specificity for linear chain recognition. Residues of NEMO involved in binding linear ubiquitin chains are required for NF-kappaB activation by TNF-alpha and other agonists, providing an explanation for the detrimental effect of NEMO mutations in patients suffering from X-linked ectodermal dysplasia and immunodeficiency.

View details for DOI 10.1016/j.cell.2009.03.007

View details for Web of Science ID 000264403900015

View details for PubMedID 19303852
The mechanism of Ca2+-dependent recognition of Alix by ALG-2: insights from X-ray crystal structures BIOCHEMICAL SOCIETY TRANSACTIONS Suzuki, H., Kawasaki, M., Inuzuka, T., Okumura, M., Kakiuchi, T., Shibata, H., Wakatsuki, S., Maki, M. 2009; 37: 190-194
Abstract

Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X] was originally identified as a protein that interacts with ALG-2, a member of the penta-EF-hand Ca(2+)-binding protein family. ALG-2 binds to its C-terminal proline-rich region that contains four tandem repeats of PXY (where X represents an uncharged amino acid). Recent X-ray crystal structural analyses of the Ca(2+)-free and Ca(2+)-bound forms of ALG-2, as well as the complex with an Alix oligopeptide, have revealed a mechanism of Ca(2+)-dependent binding of ALG-2 to its target protein. Binding of Ca(2+) to EF3 (third EF-hand) enables the side chain of Arg(125), present in the loop connecting EF3 and EF4 (fourth EF-hand), to move sufficiently to make a primary hydrophobic pocket accessible to the critical PPYP (Pro-Pro-Tyr-Pro) motif in Alix, which partially overlaps with the GPP (Gly-Pro-Pro) motif for binding to Cep55 (centrosome protein of 55 kDa). The fact that ALG-2 forms a homodimer and each monomer has one peptide-binding site indicates the possibility that ALG-2 bridges two interacting proteins, including Alix and Tsg101 (tumour susceptibility gene 101), and functions as a Ca(2+)-dependent adaptor protein.

View details for DOI 10.1042/BST0370190

View details for Web of Science ID 000263017400038

View details for PubMedID 19143629
Structural analysis of the recognition mechanism of poly-N-acetyllactosamine by the human galectin-9 N-terminal carbohydrate recognition domain GLYCOBIOLOGY Nagae, M., Nishi, N., Murata, T., Usui, T., Nakamura, T., Wakatsuki, S., Kato, R. 2009; 19 (2): 112-117
Abstract

Galectins are a family of beta-galactoside-specific lectins bearing a conserved carbohydrate recognition domain. Interactions between galectins and poly-N-acetyllactosamine sequences are critical in a variety of biological processes. Galectin-9, a member of the galectin family, has two carbohydrate recognition domains at both the N- and C-terminal regions. Here we report the crystal structure of the human galectin-9 N-terminal carbohydrate recognition domain in complex with N-acetyllactosamine dimers and trimers. These complex structures revealed that the galectin-9 N-terminal carbohydrate recognition domain can recognize internal N-acetyllactosamine units within poly-N-acetyllactosamine chains. Based on these complex structures, we propose two putative recognition modes for poly-N-acetyllactosamine binding by galectins.

View details for DOI 10.1093/glycob/cwn121

View details for Web of Science ID 000261997000002

View details for PubMedID 18977853
[Structural biology of vesicular transport]. Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme Kawasaki, M., Wakatsuki, S. 2008; 53 (16): 2071-2077
View details for PubMedID 21038587
Nucleotide-Dependent Conformational Changes and Assembly of the AAA ATPase SKD1/VPS4B TRAFFIC Inoue, M., Kamikubo, H., Kataoka, M., Kato, R., Yoshimori, T., Wakatsuki, S., Kawasaki, M. 2008; 9 (12): 2180-2189
Abstract

SKD1/VPS4B belongs to the adenosine triphosphatases associated with diverse cellular activities (AAA) family and regulates multivesicular body (MVB) biogenesis. SKD1 changes its oligomeric state during the ATPase cycle and subsequently releases endosomal sorting complex required for transport (ESCRT) complexes from endosomes during the formation of MVBs. In this study, we describe domain motions in monomeric SKD1 on ATP and ADP binding. Nucleotides bind between the alpha/beta and the alpha-helical domains of SKD1, inducing a approximately 20 degrees domain rotation and closure of the binding site, which are similar to the changes observed in the AAA+ ATPase, HslU. Gel filtration and small-angle X-ray scattering experiments showed that the ATP-bound form of SKD1 oligomerizes in solution, whereas ADP-bound and apo forms of SKD1 exist as monomers, even though the conformations of the ADP- and ATP-bound forms are nearly identical. Nucleotide-bound SKD1 structures are compatible with a hexameric ring arrangement reminiscent of the AAA ATPase p97 D1 ring. In the hexameric ring model of SKD1, Arg290 from a neighboring molecule binds to the gamma-phosphate of ATP, which promotes oligomerization of the ATP-bound form. ATP hydrolysis would eliminate this interaction and subsequent nucleotide release causes the domains to rotate, which together lead to the disassembly of the SKD1 oligomer.

View details for DOI 10.1111/j.1600-0854.2008.00831.x

View details for Web of Science ID 000261086000015

View details for PubMedID 18796009
1,2-alpha-L-Fucosynthase: A glycosynthase derived from an inverting alpha-glycosidase with an unusual reaction mechanism FEBS LETTERS Wada, J., Honda, Y., Nagae, M., Kato, R., Wakatsuki, S., Katayama, T., Taniguchi, H., Kumagai, H., Kitaoka, M., Yamamoto, K. 2008; 582 (27): 3739-3743
Abstract

Fucosyloligosaccharides have great therapeutic potential. Here we present a new route for synthesizing a Fucalpha1,2Gal linkage by introducing glycosynthase technology into 1,2-alpha-l-fucosidase. The enzyme adopts a unique reaction mechanism, in which asparagine-423 activated by aspartic acid-766 acts as a base while asparagine-421 fixes both a catalytic water and glutamic acid-566 (an acid) in the proper orientations. Glycosynthase activity of N421G, N423G, and D766G mutants was examined using beta-fucosyl fluoride and lactose, and among them, the D766G mutant most effectively synthesized 2'-fucosyllactose. 1,2-alpha-l-Fucosynthase is the first glycosynthase derived from an inverting alpha-glycosidase and from a glycosidase with an unusual reaction mechanism.

View details for DOI 10.1016/j.febslet.2008.09.054

View details for Web of Science ID 000261234800006

View details for PubMedID 18845150
Crystallization and X-ray diffraction analysis of N-terminally truncated human ALG-2 ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS Suzuki, H., Kawasaki, M., Kakiuchi, T., Shibata, H., Wakatsuki, S., Maki, M. 2008; 64: 974-977
Abstract

ALG-2 (apoptosis-linked gene 2) is an apoptosis-linked calcium-binding protein with five EF-hand motifs in the C-terminal region. N-terminally truncated ALG-2 (des3-23ALG-2) was crystallized by the vapour-diffusion method in buffer consisting of either 50 mM MES pH 6.5, 12.5%(v/v) 2-propanol and 150 mM calcium acetate or 100 mM MES pH 6.0, 15%(v/v) ethanol and 200 mM zinc acetate. Crystals of the Ca(2+)-bound form belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 54.8, b = 154.4, c = 237.7 A, alpha = beta = gamma = 90 degrees , and diffracted to 3.1 A resolution. Crystals of the Zn(2+)-bound form belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 52.8, b = 147.5, c = 230.7 A, alpha = beta = gamma = 90 degrees , and diffracted to 3.3 A resolution. The structures of the Ca(2+)-bound form and the Zn(2+)-bound form were solved by the molecular-replacement method. Although both crystals contained eight ALG-2 molecules per asymmetric unit, the metal-ion locations and octameric arrangements were found to be significantly different.

View details for DOI 10.1107/S1744309108030297

View details for Web of Science ID 000260594900002

View details for PubMedID 18997320
Structural Basis for Ca2+-Dependent Formation of ALG-2/Alix Peptide Complex: Ca2+/EF3-Driven Arginine Switch Mechanism STRUCTURE Suzuki, H., Kawasaki, M., Inuzuka, T., Okumura, M., Kakiuchi, T., Shibata, H., Wakatsuki, S., Maki, M. 2008; 16 (10): 1562-1573
Abstract

ALG-2 belongs to the penta-EF-hand (PEF) protein family and interacts with various intracellular proteins, such as Alix and TSG101, that are involved in endosomal sorting and HIV budding. Through X-ray crystallography, we solved the structures of Ca(2+)-free and -bound forms of N-terminally truncated human ALG-2 (des3-20ALG-2), Zn(2+)-bound form of full-length ALG-2, and the structure of the complex between des3-23ALG-2 and the peptide corresponding to Alix799-814 in Zn(2+)-bound form. Binding of Ca(2+) to EF3 enables the side chain of Arg125, present in the loop connecting EF3 and EF4, to move enough to make a primary hydrophobic pocket accessible to the critical PPYP motif, which partially overlaps with the GPP motif for the binding of Cep55 (centrosome protein 55 kDa). Based on these results, together with the results of in vitro binding assay with mutant ALG-2 and Alix proteins, we propose a Ca(2+)/EF3-driven arginine switch mechanism for ALG-2 binding to Alix.

View details for DOI 10.1016/j.str.2008.07.012

View details for Web of Science ID 000259930800015

View details for PubMedID 18940611
Elucidation of Rab27 Recruitment by Its Effectors: Structure of Rab27a Bound to Exophilin4/Slp2-a STRUCTURE Chavas, L. M., Ihara, K., Kawasaki, M., Torii, S., Uejima, T., Kato, R., Izumi, T., Wakatsuki, S. 2008; 16 (10): 1468-1477
Abstract

Rab GTPases coordinate vesicular trafficking within eukaryotic cells by collaborating with a set of effector proteins. Rab27a regulates numerous exocytotic pathways, and its dysfunction causes the Griscelli syndrome human immunodeficiency. Exophilin4/Slp2-a localizes on phosphatidylserine-enriched plasma membrane, and its N-terminal Rab27-binding domain (RBD27) specifically recognizes Rab27 on the surfaces of melanosomes and secretory granules prior to docking and fusion. To characterize the selective binding of Rab27 to 11 various effectors, we have determined the 1.8 A resolution structure of Rab27a in complex with Exophilin4 RBD27. The effector packs against the switch and interswitch elements of Rab27a, and specific affinity toward Rab27a is modulated by a shift in the orientation of the effector structural motif (S/T)(G/L)xW(F/Y)(2). The observed structural complementation between the interacting surfaces of Rab27a and Exophilin4 sheds light on the disparities among the Rab27 effectors and outlines a general mechanism for their recruitment.

View details for DOI 10.1016/j.str.2008.07.015

View details for Web of Science ID 000259930800006

View details for PubMedID 18940603
Biophysical methods: structure, function and dynamics studies of macromolecular assemblies using electrons, lasers, neutrons and X-rays CURRENT OPINION IN STRUCTURAL BIOLOGY Hasnain, S. S., Wakatsuki, S. 2008; 18 (5): 577-580
View details for DOI 10.1016/j.sbi.2008.09.001

View details for Web of Science ID 000260821300008

View details for PubMedID 18801436
[Molecular basis of sugar-protein interaction]. Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme Kato, R., Nagae, M., Wakatsuki, S. 2008; 53 (12): 1670-1675
View details for PubMedID 21089386
Purification, crystallization and preliminary X-ray crystallographic analysis of Rab27a GTPase in complex with exophilin4/Slp2-a effector ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS Chavas, L. M., Ihara, K., Kawasaki, M., Kato, R., Izumi, T., Wakatsuki, S. 2008; 64: 599-601
Abstract

By switching between GTP-active and GDP-inactive conformations, small Ras GTPases partly regulate membrane trafficking, cell growth and cytoskeleton dynamics. Among Rab GTPases, the Rab27 subfamily, which comprises Rab27a and Rab27b, controls the proper targeting of secretory vesicles to the plasma membrane. GppNHp-bound Rab27a in complex with the Rab27-binding domain of exophilin4/Slp2-a effector has been purified and crystallized for structural studies. The crystals belong to space group P2(1)2(1)2(1) and a complete data set was collected to a resolution of 1.8 A. Eventually, the structural characterization of the Rab27a-exophilin4/Slp2-a complex will clarify Rab27 recognition by its effectors prior to vesicle tethering and docking.

View details for DOI 10.1107/S1744309108009251

View details for Web of Science ID 000257249000007

View details for PubMedID 18607085
Structural basis for tropomyosin overlap in thin (actin) filaments and the generation of a molecular swivel by troponin-T PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Murakami, K., Stewart, M., Nozawa, K., Tomii, K., Kudou, N., Igarashi, N., Shirakihara, Y., Wakatsuki, S., Yasunaga, T., Wakabayashi, T. 2008; 105 (20): 7200-7205
Abstract

Head-to-tail polymerization of tropomyosin is crucial for its actin binding, function in actin filament assembly, and the regulation of actin-myosin contraction. Here, we describe the 2.1 A resolution structure of crystals containing overlapping tropomyosin N and C termini (TM-N and TM-C) and the 2.9 A resolution structure of crystals containing TM-N and TM-C together with a fragment of troponin-T (TnT). At each junction, the N-terminal helices of TM-N were splayed, with only one of them packing against TM-C. In the C-terminal region of TM-C, a crucial water in the coiled-coil core broke the local 2-fold symmetry and helps generate a kink on one helix. In the presence of a TnT fragment, the asymmetry in TM-C facilitates formation of a 4-helix bundle containing two TM-C chains and one chain each of TM-N and TnT. Mutating the residues that generate the asymmetry in TM-C caused a marked decrease in the affinity of troponin for actin-tropomyosin filaments. The highly conserved region of TnT, in which most cardiomyopathy mutations reside, is crucial for interacting with tropomyosin. The structure of the ternary complex also explains why the skeletal- and cardiac-muscle specific C-terminal region is required to bind TnT and why tropomyosin homodimers bind only a single TnT. On actin filaments, the head-to-tail junction can function as a molecular swivel to accommodate irregularities in the coiled-coil path between successive tropomyosins enabling each to interact equivalently with the actin helix.

View details for DOI 10.1073/pnas.0801950105

View details for Web of Science ID 000256162900019

View details for PubMedID 18483193
High-throughput operation of sample-exchange robots with double tongs at the Photon Factory beamlines JOURNAL OF SYNCHROTRON RADIATION Hiraki, M., Watanabe, S., Phonda, N., Yamada, Y., Matsugaki, N., Igarashi, N., Gaponov, Y., Wakatsuki, S. 2008; 15: 300-303
Abstract

Sample-exchange robots that can exchange cryo-pins bearing protein crystals out of experimental hutches according to user instructions have been developed. The robots were designed based on the SAM (Stanford Synchrotron Research Laboratory automated mounting) system. In order to reduce the time required for the sample exchange, the single tongs of the SAM system were modified and a double-tongs system that can hold two cryo-pins at the same time was developed. Robots with double tongs can move to the goniometer head holding the next cryo-pin with one set of tongs, dismount the experimented cryo-pin with the other set, and then mount the next pin onto the goniometer head without leaving the diffractometer area. Two different types of tongs have been installed: single tongs at beamlines BL-5A and AR-NW12A, and a double-tongs system at beamline BL-17A of the Photon Factory. The same graphical user interface software for operation of the sample-exchange robots is used at all beamlines, however, so that users do not need to consider differences between the systems. In a trial, the robot with double tongs could exchange samples within 10 s.

View details for DOI 10.1107/S0909049507064680

View details for Web of Science ID 000255049900028

View details for PubMedID 18421164
Development of an X-ray HARP-FEA detector system for high-throughput protein crystallography JOURNAL OF SYNCHROTRON RADIATION Miyoshi, T., Igarashi, N., Matsugaki, N., Yamada, Y., Hirano, K., Hyodo, K., Tanioka, K., Egami, N., Namba, M., Kubota, M., Kawai, T., Wakatsuki, S. 2008; 15: 281-284
Abstract

A new detector system for protein crystallography is now being developed based on an X-ray HARP-FEA (high-gain avalanche rushing amorphous photoconductor-field emitter array), which consists of an amorphous selenium membrane and a matrix field emitter array. The combination of the membrane avalanche effect with a single driven FEA has several advantages over currently available area detectors, including higher sensitivity, higher spatial resolution and a higher frame rate. Preliminary evaluation of the detector has been carried out and its effectiveness has been confirmed. Next, diffraction images were measured with continuous rotation of a protein crystal, and the images were compared with those measured by the existing CCD detector; the system successfully obtained high-spatial-resolution images. Using shutterless measurement, the total measurement time can be reduced significantly, making the method appropriate for high-throughput protein crystallography. The X-ray HARP-FEA detector is an attractive candidate for the next generation of X-ray area detectors.

View details for DOI 10.1107/S0909049508006584

View details for Web of Science ID 000255049900023

View details for PubMedID 18421159
Miranda cargo-binding domain forms an elongated coiled-coil homodimer in solution: Implications for asymmetric cell division in Drosophila PROTEIN SCIENCE Yousef, M. S., Kamikubo, H., Kataoka, M., Kato, R., Wakatsuki, S. 2008; 17 (5): 908-917
Abstract

Miranda is a multidomain adaptor protein involved in neuroblast asymmetric division in Drosophila melanogaster. The central domain of Miranda is necessary for cargo binding of the neural transcription factor Prospero, the Prospero-mRNA carrier Staufen, and the tumor suppressor Brat. Here, we report the first solution structure of Miranda central "cargo-binding" domain (residues 460-660) using small-angle X-ray scattering. Ab initio modeling of the scattering data yields an elongated "rod-like" molecule with a maximum linear dimension (D(max)) of approximately 22 nm. Moreover, circular dichroism and cross-linking experiments indicate that the cargo-binding domain is predominantly helical and forms a parallel coiled-coil homodimer in solution. Based on the results, we modeled the full-length Miranda protein as a double-headed, double-tailed homodimer with a long central coiled-coil region. We discuss the cargo-binding capacity of the central domain and propose a structure-based mechanism for cargo release and timely degradation of Miranda in developing neuroblasts.

View details for DOI 10.1110/ps.083431408

View details for Web of Science ID 000255290600012

View details for PubMedID 18369190
X-ray beam stabilization at BL-17A, the protein microcrystallography beamline of the Photon Factory JOURNAL OF SYNCHROTRON RADIATION Igarashi, N., Ikuta, K., Miyoshi, T., Matsugaki, N., Yamada, Y., Yousef, M. S., Wakatsuki, S. 2008; 15: 292-295
Abstract

BL-17A is a new structural biology beamline at the Photon Factory, Japan. The high-brilliance beam, derived from the new short-gap undulator (SGU#17), allows for unique protein crystallographic experiments such as data collection from microcrystals and structural determination using softer X-rays. However, microcrystal experiments require robust beam stability during data collection and minor fluctuations could not be ignored. Initially, significant beam instability was observed at BL-17A. The causes of the beam instability were investigated and its various sources identified. Subsequently, several effective countermeasures have been implemented, and the fluctuation of the beam intensity successfully suppressed to within 1%. Here the instability reduction techniques used at BL-17A are presented.

View details for DOI 10.1107/S0909049507067118

View details for Web of Science ID 000255049900026

View details for PubMedID 18421162
Implementation of remote monitoring and diffraction evaluation systems at the Photon Factory macromolecular crystallography beamlines JOURNAL OF SYNCHROTRON RADIATION Yamada, Y., Phonda, N., Matsugaki, N., Igarashi, N., Hiraki, M., Wakatsuki, S. 2008; 15: 296-299
Abstract

Owing to recent advances in high-throughput technology in macromolecular crystallography beamlines, such as high-brilliant X-ray sources, high-speed readout detectors and robotics, the number of samples that can be examined in a single visit to the beamline has increased dramatically. In order to make these experiments more efficient, two functions, remote monitoring and diffraction image evaluation, have been implemented in the macromolecular crystallography beamlines at the Photon Factory (PF). Remote monitoring allows scientists to participate in the experiment by watching from their laboratories, without having to come to the beamline. Diffraction image evaluation makes experiments easier, especially when using the sample exchange robot. To implement these two functions, two independent clients have been developed that work specifically for remote monitoring and diffraction image evaluation. In the macromolecular crystallography beamlines at PF, beamline control is performed using STARS (simple transmission and retrieval system). The system adopts a client-server style in which client programs communicate with each other through a server process using the STARS protocol. This is an advantage of the extension of the system; implementation of these new functions required few modifications of the existing system.

View details for DOI 10.1107/S0909049508004019

View details for Web of Science ID 000255049900027

View details for PubMedID 18421163
[Structural and functional biology for post translational modification and transport in eukaryotes]. Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme Wakatsuki, S., Kato, R., Kawasaki, M., Igarashi, N., Masahiko, H. 2008; 53 (5): 628-631
View details for PubMedID 18409553
Structural basis for specific lipid recognition by CERT responsible for nonvesicular trafficking of ceramide PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kudo, N., Kurnagai, K., Tornishige, N., Yamaji, T., Wakatsulki, S., Nishijima, M., Hanada, K., Kato, R. 2008; 105 (2): 488-493
Abstract

In mammalian cells, ceramide is synthesized in the endoplasmic reticulum and transferred to the Golgi apparatus for conversion to sphingomyelin. Ceramide transport occurs in a nonvesicular manner and is mediated by CERT, a cytosolic 68-kDa protein with a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. The CERT START domain efficiently transfers natural D-erythro-C16-ceramide, but not lipids with longer (C20) amide-acyl chains. The molecular mechanisms of ceramide specificity, both stereo-specific recognition and length limit, are not well understood. Here we report the crystal structures of the CERT START domain in its apo-form and in complex with ceramides having different acyl chain lengths. In these complex structures, one ceramide molecule is buried in a long amphiphilic cavity. At the far end of the cavity, the amide and hydroxyl groups of ceramide form a hydrogen bond network with specific amino acid residues that play key roles in stereo-specific ceramide recognition. At the head of the ceramide molecule, there is no extra space to accommodate additional bulky groups. The two aliphatic chains of ceramide are surrounded by the hydrophobic wall of the cavity, whose size and shape dictate the length limit for cognate ceramides. Furthermore, local high-crystallographic B-factors suggest that the alpha-3 and the Omega1 loop might work as a gate to incorporate the ceramide into the cavity. Thus, the structures demonstrate the structural basis for the mechanism by which CERT can distinguish ceramide from other lipid types yet still recognize multiple species of ceramides.

View details for DOI 10.1073/pnas.0709191105

View details for Web of Science ID 000252551100019

View details for PubMedID 18184806
Structural analysis of the human galectin-9 N-terminal carbohydrate recognition domain reveals unexpected properties that differ from the mouse orthologue JOURNAL OF MOLECULAR BIOLOGY Nagae, M., Nishi, N., Nakamura-Tsuruta, S., Hirabayashi, J., Wakatsuki, S., Kato, R. 2008; 375 (1): 119-135
Abstract

Galectins are a family of beta-galactoside-binding lectins that contain a conserved carbohydrate recognition domain (CRD). They exhibit high affinities for small beta-galactosides as well as variable binding specificities for complex glycoconjugates. Structural and biochemical analyses of the mechanism governing specific carbohydrate recognition provide a useful template to elucidate the function of these proteins. Here we report the crystal structures of the human galectin-9 N-terminal CRD (NCRD) in the presence of lactose and Forssman pentasaccharide. Mouse galectin-9 NCRD, the structure of which was previously solved by our group, forms a non-canonical dimer in both the crystal state and in solution. Human galectin-9 NCRD, however, exists as a monomer in crystals, despite a high sequence identity to the mouse homologue. Comparative frontal affinity chromatography analysis of the mouse and human galectin-9 NCRDs revealed different carbohydrate binding specificities, with disparate affinities for complex glycoconjugates. Human galectin-9 NCRD exhibited a high affinity for Forssman pentasaccharide; the association constant for mouse galectin-9 NCRD was 100-fold less than that observed for the human protein. The combination of structural data with mutational studies demonstrated that non-conserved amino acid residues on the concave surface were important for determination of target specificities. The human galectin-9 NCRD exhibited greater inhibition of cell proliferation than the mouse NCRD. We discuss the biochemical and structural differences between highly homologous proteins from different species.

View details for DOI 10.1016/j.jmb.2007.09.060

View details for Web of Science ID 000252002100011

View details for PubMedID 18005988
Design of disulfide-linked thioredoxin dimers and multimers through analysis of crystal contacts JOURNAL OF MOLECULAR BIOLOGY Das, M., Kobayashi, M., Yamada, Y., Sreeramulu, S., Ramakrishnan, C., Wakatsuki, S., Kato, R., Varadarajan, R. 2007; 372 (5): 1278-1292
Abstract

Disulfide bonds play an important role in protein stability and function. Here, we describe a general procedure for generating disulfide-linked dimers and multimers of proteins of known crystal structures. An algorithm was developed to predict sites in a protein compatible with intermolecular disulfide formation with neighboring molecules in the crystal lattice. A database analysis was carried out on 46 PDB coordinates to verify the general applicability of this algorithm to predict intermolecular disulfide linkages. On the basis of the predictions from this algorithm, mutants were constructed and characterized for a model protein, thioredoxin. Of the five mutants, as predicted, in solution four formed disulfide-linked dimers while one formed polymers. Thermal and chemical denaturation studies on these mutant thioredoxins showed that three of the four dimeric mutants had similar stability to wild-type thioredoxin while one had lower stability. Three of the mutant dimers crystallized readily (in four to seven days) in contrast to the wild-type protein, which is particularly difficult to crystallize and takes more than a month to form diffraction-quality crystals. In two of the three cases, the structure of the dimer was exactly as predicted by the algorithm, while in the third case the relative orientation of the monomers in the dimer was different from the predicted one. This methodology can be used to enhance protein crystallizability, modulate the oligomerization state and to produce linear chains or ordered three-dimensional protein arrays.

View details for DOI 10.1016/j.jmb.2007.07.033

View details for Web of Science ID 000249817500012

View details for PubMedID 17727880
Structural basis for recognition of high mannose type glycoproteins by mammalian transport lectin VIP36 JOURNAL OF BIOLOGICAL CHEMISTRY Satoh, T., Cowieson, N. P., Hakamata, W., Ideo, H., Fukushima, K., Kurihara, M., Kato, R., Yamashita, K., Wakatsuki, S. 2007; 282 (38): 28246-28255
Abstract

VIP36 functions as a transport lectin for trafficking certain high mannose type glycoproteins in the secretory pathway. Here we report the crystal structure of VIP36 exoplasmic/luminal domain comprising a carbohydrate recognition domain and a stalk domain. The structures of VIP36 in complex with Ca(2+) and mannosyl ligands are also described. The carbohydrate recognition domain is composed of a 17-stranded antiparallel beta-sandwich and binds one Ca(2+) adjoining the carbohydrate-binding site. The structure reveals that a coordinated Ca(2+) ion orients the side chains of Asp(131), Asn(166), and His(190) for carbohydrate binding. This result explains the Ca(2+)-dependent carbohydrate binding of this protein. The Man-alpha-1,2-Man-alpha-1,2-Man, which corresponds to the D1 arm of high mannose type glycan, is recognized by eight residues through extensive hydrogen bonds. The complex structures reveal the structural basis for high mannose type glycoprotein recognition by VIP36 in a Ca(2+)-dependent and D1 arm-specific manner.

View details for DOI 10.1074/jbc.M703064200

View details for Web of Science ID 000249455600074

View details for PubMedID 17652092
Molecular basis for autoregulatory interaction between GAE domain and hinge region of GGA1 TRAFFIC Inoue, M., Shiba, T., Ihara, K., Yamada, Y., Hirano, S., Kamikubo, H., Kataoka, M., Kawasaki, M., Kato, R., Nakayama, K., Wakatsuki, S. 2007; 8 (7): 904-913
Abstract

Golgi-localizing, gamma-adaptin ear domain homology, ADP ribosylation factor-binding (GGA) proteins and the adaptor protein (AP) complex, AP-1, are involved in membrane traffic between the trans Golgi network and the endosomes. The gamma-adaptin ear (GAE) domain of GGAs and the gamma1 ear domain of AP-1 interact with an acidic phenylalanine motif found in accessory proteins. The GAE domain of GGA1 (GGA1-GAE) interacts with a WNSF-containing peptide derived from its own hinge region, although the peptide sequence deviates from the standard acidic phenylalanine motif. We report here the structure of GGA1-GAE in complex with the GGA1 hinge peptide, which revealed that the two aromatic side chains of the WNSF sequence fit into a hydrophobic groove formed by aliphatic portions of the side chains of conserved arginine and lysine residues of GGA1-GAE, in a similar manner to the interaction between GGA-GAEs and acidic phenylalanine sequences from the accessory proteins. Fluorescence quenching experiments indicate that the GGA1 hinge region binds to GGA1-GAE and competes with accessory proteins for binding. Taken together with the previous observation that gamma1 ear binds to the GGA1 hinge region, the interaction between the hinge region and the GAE domain underlies the autoregulation of GGA function in clathrin-mediated trafficking through competing with the accessory proteins and the AP-1 complex.

View details for DOI 10.1111/j.1600-0854.2007.00577.x

View details for Web of Science ID 000247440800010

View details for PubMedID 17506864
Structure of the small GTPase Rab27b shows an unexpected swapped dimer ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Chavas, L. M., Torii, S., Kamikubo, H., Kawasaki, M., Ihara, K., Kato, R., Kataoka, M., Izumi, T., Wakatsuki, S. 2007; 63: 769-779
Abstract

Members of the Rab family of small GTPases regulate membrane traffic within the cell by recruiting their specific effectors in a nucleotide-dependent manner. The Rab27 subfamily consists of Rab27a and Rab27b, which share 70% sequence identity. By interacting with a large set of effector proteins such as melanophilin and granuphilin, both Rab27a and Rab27b regulate the exocytosis of secretory lysosomes. Here, the crystal structures of mouse Rab27b in complex with GDP have been determined in three distinct crystal lattices. Surprisingly, Rab27b-GDP exists in an open conformation with protruding switch and interswitch regions, which are stabilized through dimerization by means of domain-swapping in the crystals. In contrast, small-angle X-ray scattering measurements showed an extended monomer form of Rab27b in solution. The observed dimer formation of Rab27b-GDP in the crystals would restrain the highly flexible switch regions. Possible biological implications of this atypical structure of Rab27b and its plausible influence in effector interaction are discussed.

View details for DOI 10.1107/S0907444907019725

View details for Web of Science ID 000247981100003

View details for PubMedID 17582168
Structural basis of the catalytic reaction mechanism of novel 1,2-alpha-L-fucosidase from Bifidobacterium bifidum JOURNAL OF BIOLOGICAL CHEMISTRY Nagae, M., Tsuchiya, A., Katayama, T., Yamamoto, K., Wakatsuki, S., Kato, R. 2007; 282 (25): 18497-18509
Abstract

1,2-alpha-L-fucosidase (AfcA), which hydrolyzes the glycosidic linkage of Fucalpha1-2Gal via an inverting mechanism, was recently isolated from Bifidobacterium bifidum and classified as the first member of the novel glycoside hydrolase family 95. To better understand the molecular mechanism of this enzyme, we determined the x-ray crystal structures of the AfcA catalytic (Fuc) domain in unliganded and complexed forms with deoxyfuconojirimycin (inhibitor), 2'-fucosyllactose (substrate), and L-fucose and lactose (products) at 1.12-2.10 A resolution. The AfcA Fuc domain is composed of four regions, an N-terminal beta region, a helical linker, an (alpha/alpha)6 helical barrel domain, and a C-terminal beta region, and this arrangement is similar to bacterial phosphorylases. In the complex structures, the ligands were buried in the central cavity of the helical barrel domain. Structural analyses in combination with mutational experiments revealed that the highly conserved Glu566 probably acts as a general acid catalyst. However, no carboxylic acid residue is found at the appropriate position for a general base catalyst. Instead, a water molecule stabilized by Asn423 in the substrate-bound complex is suitably located to perform a nucleophilic attack on the C1 atom of L-fucose moiety in 2'-fucosyllactose, and its location is nearly identical near the O1 atom of beta-L-fucose in the products-bound complex. Based on these data, we propose and discuss a novel catalytic reaction mechanism of AfcA.

View details for DOI 10.1074/jbc.M701534200

View details for Web of Science ID 000247302000055

View details for PubMedID 17459873
X-ray phase imaging of biological soft tissue using a direct-sensing x-ray HARP tube camera PHYSICS IN MEDICINE AND BIOLOGY Hirano, K., Miyoshi, T., Igarashi, N., Takeda, T., Wu, J., Lwin, T., Kubota, M., Egami, N., Tanioka, K., Kawai, T., Wakatsuki, S. 2007; 52 (9): 2545-2552
Abstract

A HDTV camera having a direct-sensing x-ray high-gain avalanche rushing amorphous photoconductor (HARP) tube was used, for the first time, to acquire x-ray phase maps. The tube can achieve a high sensitivity as a result of the avalanche multiplication process in the HARP target. A beryllium plate, rather than a glass plate, was used as the face plate of the tube to minimize the loss of x-rays due to absorption, and a 15 microm thick HARP target was directly formed on it. In the experiment, the x-ray phase shifts produced by a rat liver were measured using synchrotron x-rays (lambda = 0.0766 nm) and a triple Laue-case (LLL) x-ray interferometer. Interference patterns produced by the sample were observed with the direct-sensing x-ray HARP tube camera. A voltage of 1300 V was applied to the HARP target to give an output signal gain of two. The camera was operated in 1125 scanning-line mode, and real-time images were stored on a workstation at a rate of 30 images/s with an image format of 960 (H) x 1100 (V) pixels. A phase-map image of the sample was successfully obtained using the fringe scanning method and phase unwrapping. The observed phase shifts ranged from 50 degrees to 200 degrees . Trees of blood vessels in the rat liver were clearly depicted without using a contrast agent. The spatial resolution of the x-ray camera was estimated to be better than 35 microm in the vertical direction and 100 microm in the horizontal direction.

View details for DOI 10.1088/0031-9155/52/9/014

View details for Web of Science ID 000246172000014

View details for PubMedID 17440251
Development of control applications for high-throughput protein crystallography experiments SYNCHROTRON RADIATION INSTRUMENTATION, PTS 1 AND 2 Gaponov, Y. A., Matsugaki, N., Honda, N., Sasajima, K., Igarashi, N., Hiraki, M., Yamada, Y., Wakatsuki, S. 2007; 879: 1932-1935
View details for Web of Science ID 000244647900459
Design and construction of a high-speed network connecting all the protein crystallography beamlines at the photon factory SYNCHROTRON RADIATION INSTRUMENTATION, PTS 1 AND 2 Matsugaki, N., Yamada, Y., Igarashi, N., Wakatsuki, S. 2007; 879: 1936-1939
View details for Web of Science ID 000244647900460
Automated sample exchange robots for the structural biology beam lines at the photon factory SYNCHROTRON RADIATION INSTRUMENTATION, PTS 1 AND 2 Hiraki, M., Watanabe, S., Yamada, Y., Matsugaki, N., Igarashi, N., Gaponov, Y., Wakatsuki, S. 2007; 879: 1924-1927
View details for Web of Science ID 000244647900457
BL-17A, a new protein micro-crystallography beam line of the photon factory SYNCHROTRON RADIATION INSTRUMENTATION, PTS 1 AND 2 Igarashi, N., Matsugaki, N., Yamada, Y., Hiraki, M., Koyama, A., Hirano, K., Miyoshi, T., Wakatsuki, S. 2007; 879: 812-815
View details for Web of Science ID 000244647900192
Crystal structure of the galectin-9 N-terminal carbohydrate recognition domain from Mus musculus reveals the basic mechanism of carbohydrate recognition JOURNAL OF BIOLOGICAL CHEMISTRY Nagae, M., Nishi, N., Murata, T., Usui, T., Nakamura, T., Wakatsuki, S., Kato, R. 2006; 281 (47): 35884-35893
Abstract

The galectins are a family of beta-galactoside-binding animal lectins with a conserved carbohydrate recognition domain (CRD). They have a high affinity for small beta-galactosides, but binding specificity for complex glycoconjugates varies considerably within the family. The ligand recognition is essential for their proper function, and the structures of several galectins have suggested their mechanism of carbohydrate binding. Galectin-9 has two tandem CRDs with a short linker, and we report the crystal structures of mouse galectin-9 N-terminal CRD (NCRD) in the absence and the presence of four ligand complexes. All structures form the same dimer, which is quite different from the canonical 2-fold symmetric dimer seen for galectin-1 and -2. The beta-galactoside recognition mechanism in the galectin-9 NCRD is highly conserved among other galectins. In the apo form structure, water molecules mimic the ligand hydrogen-bond network. The galectin-9 NCRD can bind both N-acetyllactosamine (Galbeta1-4GlcNAc) and T-antigen (Galbeta1-3GalNAc) with the proper location of Arg-64. Moreover, the structure of the N-acetyllactosamine dimer (Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc) complex shows a unique binding mode of galectin-9. Finally, surface plasmon resonance assay showed that the galectin-9 NCRD forms a homophilic dimer not only in the crystal but also in solution.

View details for DOI 10.1074/jbc.M606648200

View details for Web of Science ID 000242100500035

View details for PubMedID 16990264
Monoubiquitylation of GGA3 by hVPS18 regulates its ubiquitin-binding ability BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Yogosawa, S., Kawasaki, M., Wakatsuki, S., Kominami, E., Shiba, Y., Nakayama, K., Kohsaka, S., Akazawa, C. 2006; 350 (1): 82-90
Abstract

GGAs (Golgi-localizing, gamma-adaptin ear domain homology, ADP-ribosylation factor (ARF)-binding proteins), constitute a family of monomeric adaptor proteins and are associated with protein trafficking from the trans-Golgi network to endosomes. Here, we show that GGA3 is monoubiquitylated by a RING-H2 type-ubiquitin ligase hVPS18 (human homologue of vacuolar protein sorting 18). By in vitro ubiquitylation assays, we have identified lysine 258 in the GAT domain as a major ubiquitylation site that resides adjacent to the ubiquitin-binding site. The ubiquitylation is abolished by a mutation in either the GAT domain or ubiquitin that disrupts the GAT-ubiquitin interaction, indicating that the ubiquitin binding is a prerequisite for the ubiquitylation. Furthermore, the GAT domain ubiquitylated by hVPS18 no longer binds to ubiquitin, indicating that ubiquitylation negatively regulates the ubiquitin-binding ability of the GAT domain. These results suggest that the ubiquitin binding and ubiquitylation of GGA3-GAT domain are mutually inseparable through a ubiquitin ligase activity of hVPS18.

View details for DOI 10.1016/j.bbrc.2006.09.013

View details for Web of Science ID 000241212700011

View details for PubMedID 16996030
Crystal structure of GlcAT-S, a human glucuronyltransferase, involved in the biosynthesis of the HNK-1 carbohydrate epitope PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS Shiba, T., Kakuda, S., Ishiguro, M., Morita, I., Oka, S., Kawasaki, T., Wakatsuki, S., Kato, R. 2006; 65 (2): 499-508
Abstract

The HNK-1 carbohydrate epitope is found in various neural cell adhesion molecules. Two glucuronyltransferases (GlcAT-P and GlcAT-S) are involved in the biosynthesis of HNK-1 carbohydrate. Our previous study on the crystal structure of GlcAT-P revealed the reaction and substrate recognition mechanisms of this enzyme. Comparative analyses of the enzymatic activities of GlcAT-S and GlcAT-P showed that there are notable differences in the acceptor substrate specificities of these enzymes. To elucidate differences between their specificities, we now solved the crystal structure of GlcAT-S. Residues interacting with UDP molecule, which is a part of the donor substrate, are highly conserved between GlcAT-P and GlcAT-S. On the other hand, there are some differences between these proteins in the manner they recognize their respective acceptor substrates. Phe245, one of the most important GlcAT-P residues for the recognition of acceptors, is a tryptophan in GlcAT-S. In addition, Val320, which is located on the C-terminal long loop of the neighboring molecule in the dimer and critical in the recognition of the acceptor sugar molecule by the GlcAT-P dimer, is an alanine in GlcAT-S. These differences play key roles in establishing the distinct specificity for the acceptor substrate by GlcAT-S, which is further supported by site-directed mutagenesis of GlcAT-S and a computer-aided model building of GlcAT-S/substrate complexes.

View details for DOI 10.1002/prot.21118

View details for Web of Science ID 000240748700021

View details for PubMedID 16897771
Structural basis of ubiquitin recognition by mammalian Eap45 GLUE domain NATURE STRUCTURAL & MOLECULAR BIOLOGY Hirano, S., Suzuki, N., Slagsvold, T., Kawasaki, M., Trambaiolo, D., Kato, R., Stenmark, H., Wakatsuki, S. 2006; 13 (11): 1031-1032
Abstract

ESCRT-II, a complex that sorts ubiquitinated membrane proteins to lysosomes, localizes to endosomes through interaction between the Vps36 subunit's GLUE domain and phosphatidylinositides (PIs). In yeast, a ubiquitin (Ub)-interacting NZF domain is inserted in Vps36 GLUE, whereas its mammalian counterpart, Eap45 GLUE, lacks the NZF domain. In the Eap45 GLUE-Ub complex structure, Ub binds far from the proposed PI-binding site of Eap45 GLUE, suggesting their independent binding.

View details for DOI 10.1038/nsmb1163

View details for Web of Science ID 000241817700018

View details for PubMedID 17057714
Structural basis for Rab11-dependent membrane recruitment of a family of Rab11-interacting protein 3 (FIP3)/Arfophilin-1 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shiba, T., Koga, H., Shin, H., Kawasaki, M., Kato, R., Nakayama, K., Wakatsuki, S. 2006; 103 (42): 15416-15421
Abstract

Family of Rab11-interacting protein (FIP)3/Arfophlin-1 and FIP4/Arfophilin-2 are dual effectors for Rab11 and ADP ribosylation factor (ARF)5/ARF6, which are involved in membrane delivery from recycling endosomes to the plasma membrane during cytokinesis. Here, we define the distinct C-terminal binding regions of FIP3 and FIP4 for Rab11 and ARF5/ARF6. Furthermore, we determined the crystal structure of Rab11 in complex with the Rab11-binding domain (RBD) of FIP3. The long amphiphilic alpha-helix of FIP3-RBD forms a parallel coiled-coil homodimer, with two symmetric interfaces with two Rab11 molecules. The hydrophobic side of the RBD helix is involved in homodimerization and mediates the interaction with the Rab11 switch 1 region, whereas the opposite hydrophilic side interacts with the Rab11 switch 2 and is the major factor contributing to the binding specificity. The bivalent interaction of FIP3 with Rab11 at the C terminus allows FIP3 to coordinately function with other binding partners, including ARFs.

View details for DOI 10.1073/pnas.0605357103

View details for Web of Science ID 000241476200027

View details for PubMedID 17030804
Development of an automated large-scale protein-crystallization and monitoring system for high-throughput protein-structure analyses ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Hiraki, M., Kato, R., Nagai, M., Satoh, T., Hirano, S., Ihara, K., Kudo, N., Nagae, M., Kobayashi, M., Inoue, M., Uejima, T., Oda, S., Chavas, L. M., Akutsu, M., Yamada, Y., Kawasaki, M., Matsugaki, N., Igarashi, N., Suzuki, M., Wakatsuki, S. 2006; 62: 1058-1065
Abstract

Protein crystallization remains one of the bottlenecks in crystallographic analysis of macromolecules. An automated large-scale protein-crystallization system named PXS has been developed consisting of the following subsystems, which proceed in parallel under unified control software: dispensing precipitants and protein solutions, sealing crystallization plates, carrying robot, incubators, observation system and image-storage server. A sitting-drop crystallization plate specialized for PXS has also been designed and developed. PXS can set up 7680 drops for vapour diffusion per hour, which includes time for replenishing supplies such as disposable tips and crystallization plates. Images of the crystallization drops are automatically recorded according to a preprogrammed schedule and can be viewed by users remotely using web-based browser software. A number of protein crystals were successfully produced and several protein structures could be determined directly from crystals grown by PXS. In other cases, X-ray quality crystals were obtained by further optimization by manual screening based on the conditions found by PXS.

View details for DOI 10.1107/S0907444906023821

View details for Web of Science ID 000240019700014

View details for PubMedID 16929107
Structural basis of carbohydrate transfer activity by human UDP-GaINAc: Polypeptide alpha-N-acetylgalactosaminyltransferase (pp-GaINAc-T10) JOURNAL OF MOLECULAR BIOLOGY Kubota, T., Shiba, T., Sugioka, S., Furukawa, S., Sawaki, H., Kato, R., Wakatsuki, S., Narimatsu, H. 2006; 359 (3): 708-727
Abstract

Mucin-type O-glycans are important carbohydrate chains involved in differentiation and malignant transformation. Biosynthesis of the O-glycan is initiated by the transfer of N-acetylgalactosamine (GalNAc) which is catalyzed by UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (pp-GalNAc-Ts). Here we present crystal structures of the pp-GalNAc-T10 isozyme, which has specificity for glycosylated peptides, in complex with the hydrolyzed donor substrate UDP-GalNAc and in complex with GalNAc-serine. A structural comparison with uncomplexed pp-GalNAc-T1 suggests that substantial conformational changes occur in two loops near the catalytic center upon donor substrate binding, and that a distinct interdomain arrangement between the catalytic and lectin domains forms a narrow cleft for acceptor substrates. The distance between the catalytic center and the carbohydrate-binding site on the lectin beta sub-domain influences the position of GalNAc glycosylation on GalNAc-glycosylated peptide substrates. A chimeric enzyme in which the two domains of pp-GalNAc-T10 are connected by a linker from pp-GalNAc-T1 acquires activity toward non-glycosylated acceptors, identifying a potential mechanism for generating the various acceptor specificities in different isozymes to produce a wide range of O-glycans.

View details for DOI 10.1016/j.jmb.2006.03.061

View details for Web of Science ID 000238297800016

View details for PubMedID 16650853
Structures of the carbohydrate recognition domain of Ca2+-independent cargo receptors Emp46p and Emp47p JOURNAL OF BIOLOGICAL CHEMISTRY Satoh, T., Sato, K., Kanoh, A., Yamashita, K., Yamada, Y., Igarashi, N., Kato, R., Nakano, A., Wakatsuki, S. 2006; 281 (15): 10410-10419
Abstract

Emp46p and Emp47p are type I membrane proteins, which cycle between the endoplasmic reticulum (ER) and the Golgi apparatus by vesicles coated with coat protein complexes I and II (COPI and COPII). They are considered to function as cargo receptors for exporting N-linked glycoproteins from the ER. We have determined crystal structures of the carbohydrate recognition domains (CRDs) of Emp46p and Emp47p of Saccharomyces cerevisiae, in the absence and presence of metal ions. Both proteins fold as a beta-sandwich, and resemble that of the mammalian ortholog, p58/ERGIC-53. However, the nature of metal binding is distinct from that of Ca(2+)-dependent p58/ERGIC-53. Interestingly, the CRD of Emp46p does not bind Ca(2+) ion but instead binds K(+) ion at the edge of a concave beta-sheet whose position is distinct from the corresponding site of the Ca(2+) ion in p58/ERGIC-53. Binding of K(+) ion to Emp46p appears essential for transport of a subset of glycoproteins because the Y131F mutant of Emp46p, which cannot bind K(+) ion fails to rescue the transport in disruptants of EMP46 and EMP47 genes. In contrast the CRD of Emp47p binds no metal ions at all. Furthermore, the CRD of Emp46p binds to glycoproteins carrying high mannosetype glycans and the is promoted by binding not the addition of Ca(2+) or K(+) ion in These results suggest that Emp46p can be regarded as a Ca(2+)-independent intracellular lectin at the ER exit sites.

View details for DOI 10.1074/jbc.M512258200

View details for Web of Science ID 000236594300070

View details for PubMedID 16439369
Double-sided ubiquitin binding of Hrs-UIM in endosomal protein sorting NATURE STRUCTURAL & MOLECULAR BIOLOGY Hirano, S., Kawasaki, M., Ura, H., Kato, R., Raiborg, C., Stenmark, H., Wakatsuki, S. 2006; 13 (3): 272-277
Abstract

Hrs has an essential role in sorting of monoubiquitinated receptors to multivesicular bodies for lysosomal degradation, through recognition of ubiquitinated receptors by its ubiquitin-interacting motif (UIM). Here, we present the structure of a complex of Hrs-UIM and ubiquitin at 1.7-A resolution. Hrs-UIM forms a single alpha-helix, which binds two ubiquitin molecules, one on either side. These two ubiquitin molecules are related by pseudo two-fold screw symmetry along the helical axis of the UIM, corresponding to a shift by two residues on the UIM helix. Both ubiquitin molecules interact with the UIM in the same manner, using the Ile44 surface, with equal binding affinities. Mutational experiments show that both binding sites of Hrs-UIM are required for efficient degradative protein sorting. Hrs-UIM belongs to a new subclass of double-sided UIMs, in contrast to its yeast homolog Vps27p, which has two tandem single-sided UIMs.

View details for DOI 10.1038/nsmb1051

View details for Web of Science ID 000235776900019

View details for PubMedID 16462748
Membrane recruitment of effector proteins by Arf and Rab GTPases CURRENT OPINION IN STRUCTURAL BIOLOGY Kawasaki, M., Nakayama, K., Wakatsuki, S. 2005; 15 (6): 681-689
Abstract

In their GTP-bound form, Arf and Rab family GTPases associate with distinct organelle membranes, to which they recruit specific sets of effector proteins that regulate vesicular transport. The Arf GTPases are involved in the formation of coated carrier vesicles by recruiting coat proteins. On the other hand, the Rab GTPases are involved in the tethering, docking and fusion of transport vesicles with target organelles, acting in concert with the tethering and fusion machineries. Recent structural studies of the Arf1-GGA and Rab5-Rabaptin-5 complexes, as well as other effector structures in complex with the Arf and Rab GTPases, have shed light on the mechanisms underlying the GTP-dependent membrane recruitment of these effector proteins.

View details for DOI 10.1016/j.sbi.2005.10.015

View details for Web of Science ID 000234167500012

View details for PubMedID 16289847
Structural basis for transcription inhibition by tagetitoxin NATURE STRUCTURAL & MOLECULAR BIOLOGY Vassylyev, D. G., Svetlov, V., Vassylyeva, M. N., Perederina, A., Igarashi, N., Matsugaki, N., Wakatsuki, S., Artsimovitch, I. 2005; 12 (12): 1086-1093
Abstract

Tagetitoxin (Tgt) inhibits transcription by an unknown mechanism. A structure at a resolution of 2.4 A of the Thermus thermophilus RNA polymerase (RNAP)-Tgt complex revealed that the Tgt-binding site within the RNAP secondary channel overlaps that of the stringent control effector ppGpp, which partially protects RNAP from Tgt inhibition. Tgt binding is mediated exclusively through polar interactions with the beta and beta' residues whose substitutions confer resistance to Tgt in vitro. Importantly, a Tgt phosphate, together with two active site acidic residues, coordinates the third Mg(2+) ion, which is distinct from the two catalytic metal ions. We show that Tgt inhibits all RNAP catalytic reactions and propose a mechanism in which the Tgt-bound Mg(2+) ion has a key role in stabilization of an inactive transcription intermediate. Remodeling of the active site by metal ions could be a common theme in the regulation of catalysis by nucleic acid enzymes.

View details for DOI 10.1038/nsmb1015

View details for Web of Science ID 000233774300018

View details for PubMedID 16273103
GGAs: Structure and function of a novel family of clathrin adaptors invoved in membrane trafficking between the TGN and endosomes SEIKAGAKU Nakayama, K., Wakatsuki, S. 2005; 77 (11): 1367-1381
View details for Web of Science ID 000233745000002

View details for PubMedID 16370355
Structural basis for recognition of ubiquitinated cargo by Tom1-GAT domain FEBS LETTERS Akutsu, M., Kawasaki, M., Katoh, Y., Shiba, T., Yamaguchi, Y., Kato, R., Kato, K., Nakayama, K., Wakatsuki, S. 2005; 579 (24): 5385-5391
Abstract

Tom1 (Target of Myb1) is suggested to be involved in the transport of ubiquitinated proteins, through the interaction of its GAT (GGA and Tom1) domain with ubiquitin. Here, we demonstrate that the three-helix bundle of Tom1-GAT has two ubiquitin-binding sites recognizing the hydrophobic Ile44 surface of ubiquitin. The complex crystal structure demonstrates that the first site is a hydrophobic patch on helices alpha1 and alpha2. NMR and biochemical data revealed that the N-terminal half of helix alpha3 of Tom1-GAT constitutes the second, stronger binding site. The double-sided ubiquitin binding enhances the efficiency of recognition of ubiquitinated proteins by Tom1.

View details for DOI 10.1016/j.febslet.2005.08.076

View details for Web of Science ID 000232523600022

View details for PubMedID 16199040
Structural basis of transcription inhibition by antibiotic streptolydigin MOLECULAR CELL Temiakov, D., Zenkin, N., Vassylyeva, M. N., Perederina, A., Tahirov, T. H., Kashkina, E., Savkina, M., Zorov, S., Nikiforov, V., Igarashi, N., Matsugaki, N., Wakatsuki, S., Severinov, K., Vassylyev, D. G. 2005; 19 (5): 655-666
Abstract

Streptolydigin (Stl) is a potent inhibitor of bacterial RNA polymerases (RNAPs). The 2.4 A resolution structure of the Thermus thermophilus RNAP-Stl complex showed that, in full agreement with the available genetic data, the inhibitor binding site is located 20 A away from the RNAP active site and encompasses the bridge helix and the trigger loop, two elements that are considered to be crucial for RNAP catalytic center function. Structure-based biochemical experiments revealed additional determinants of Stl binding and demonstrated that Stl does not affect NTP substrate binding, DNA translocation, and phosphodiester bond formation. The RNAP-Stl complex structure, its comparison with the closely related substrate bound eukaryotic transcription elongation complexes, and biochemical analysis suggest an inhibitory mechanism in which Stl stabilizes catalytically inactive (preinsertion) substrate bound transcription intermediate, thereby blocking structural isomerization of RNAP to an active configuration. The results provide a basis for a design of new antibiotics utilizing the Stl-like mechanism.

View details for DOI 10.1016/j.molcel.2005.07.020

View details for Web of Science ID 000231692900007

View details for PubMedID 16167380
Allosteric modulation of the RNA polymerase catalytic reaction is an essential component of transcription control by rifamycins CELL Artsimovitch, I., Vassylyeva, M. N., Svetlov, D., Svetlov, V., Perederina, A., Igarashi, N., Matsugaki, N., Wakatsuki, S., Tahirov, T. H., Vassylyev, D. G. 2005; 122 (3): 351-363
Abstract

Rifamycins, the clinically important antibiotics, target bacterial RNA polymerase (RNAP). A proposed mechanism in which rifamycins sterically block the extension of nascent RNA beyond three nucleotides does not alone explain why certain RNAP mutations confer resistance to some but not other rifamycins. Here we show that unlike rifampicin and rifapentin, and contradictory to the steric model, rifabutin inhibits formation of the first and second phosphodiester bonds. We report 2.5 A resolution structures of rifabutin and rifapentin complexed with the Thermus thermophilus RNAP holoenzyme. The structures reveal functionally important distinct interactions of antibiotics with the initiation sigma factor. Strikingly, both complexes lack the catalytic Mg2+ ion observed in the apo-holoenzyme, whereas an increase in Mg2+ concentration confers resistance to rifamycins. We propose that a rifamycin-induced signal is transmitted over approximately 19 A to the RNAP active site to slow down catalysis. Based on structural predictions, we designed enzyme substitutions that apparently interrupt this allosteric signal.

View details for DOI 10.1016/j.cell.2005.07.014

View details for Web of Science ID 000231254400008

View details for PubMedID 16096056
Structure of the ectodomain of Drosophila peptidoglycan-recognition protein LCa suggests a molecular mechanism for pattern recognition PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chang, C. I., Ihara, K., Chelliah, Y., Mengin-Lecreulx, D., Wakatsuki, S., Deisenhofer, J. 2005; 102 (29): 10279-10284
Abstract

The peptidoglycan-recognition protein LCa (PGRP-LCa) is a transmembrane receptor required for activation of the Drosophila immune deficiency pathway by monomeric Gram-negative peptidoglycan. We have determined the crystal structure of the ectodomain of PGRP-LCa at 2.5-A resolution and found two unique helical insertions in the LCa ectodomain that disrupt an otherwise L-shaped peptidoglycan-docking groove present in all other known PGRP structures. The deficient binding of PGRP-LCa to monomeric peptidoglycan was confirmed by biochemical pull-down assays. Recognition of monomeric peptidoglycan involves both PGRP-LCa and -LCx. We showed that association of the LCa and LCx ectodomains in vitro depends on monomeric peptidoglycan. The presence of a defective peptidoglycan-docking groove, while preserving a unique role in mediating monomeric peptidoglycan induction of immune response, suggests that PGRP-LCa recognizes the exposed structural features of a monomeric muropeptide when the latter is bound to and presented by the ectodomain of PGRP-LCx. Such features include N-acetyl glucosamine and the anhydro bond in the glycan of the muropeptide, which have been demonstrated to be critical for immune stimulatory activity.

View details for DOI 10.1073/pnas.0504547102

View details for Web of Science ID 000230665800053

View details for PubMedID 16006509
Molecular mechanism of ubiquitin recognition by GGA3 GAT domain GENES TO CELLS Kawasaki, M., Shiba, T., Shiba, Y., Yamaguchi, Y., Matsugaki, N., Igarashi, N., Suzuki, M., Kato, R., Kato, K., Nakayama, K., Wakatsuki, S. 2005; 10 (7): 639-654
Abstract

GGA (Golgi-localizing, gamma-adaptin ear domain homology, ARF-binding) proteins, which constitute a family of clathrin coat adaptor proteins, have recently been shown to be involved in the ubiquitin-dependent sorting of receptors, through the interaction between the C-terminal three-helix-bundle of the GAT (GGA and Tom1) domain (C-GAT) and ubiquitin. We report here the crystal structure of human GGA3 C-GAT in complex with ubiquitin. A hydrophobic patch on C-GAT helices alpha1 and alpha2 forms a binding site for the hydrophobic Ile44 surface of ubiquitin. Two distinct orientations of ubiquitin Arg42 determine the shape and the charge distribution of ubiquitin Ile44 surface, leading to two different binding modes. Biochemical and NMR data strongly suggest another hydrophobic binding site on C-GAT helices alpha2 and alpha3, opposite to the first binding site, also binds ubiquitin although weakly. The double-sided ubiquitin binding provides the GAT domain with higher efficiency in recognizing ubiquitinated receptors for lysosomal receptor degradation.

View details for DOI 10.1111/j.1365-2443.2005.00865.x

View details for Web of Science ID 000229913200002

View details for PubMedID 15966896
Structure determination of GGA-GAE and gamma 1-ear in complex with peptides: crystallization of low-affinity complexes in membrane traffic ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY Yamada, Y., Inoue, M., Shiba, T., Kawasaki, M., Kato, R., Nakayama, K., Wakatsuki, S. 2005; 61: 731-736
Abstract

Crystallization of protein-protein complexes is an important step in the studies of biological functions of proteins. However, weak and transient, even though specific, interactions often present difficulties in crystallization of protein complexes due to the heterogeneity of the sample mixture. For example, the gamma1-ear domain of the AP-1 complex and the GAE domain of GGA1, responsible for the interaction with accessory proteins involved in vesicular transport, are known to interact with target proteins with affinities of the order of 1-100 microM. Such low affinities have hampered crystallization trials of the complexes. To overcome this problem, the gamma1-ear and GAE domains were first co-crystallized with excess amounts of the peptides. Co-crystals of both domains were obtained and the complex structures were determined at 2.5-2.9 A resolution. Based on the crystal packing of gamma1-ear and the cognate peptide, gamma1-ear fused with a peptide tag at the N-terminus was prepared. The peptide-tagged gamma1-ear readily crystallized and the crystal diffracted far better, 1.9-2.2 A resolution, compared with the co-crystallized complex, giving significantly more details without affecting the overall complex structure.

View details for DOI 10.1107/S0907444905009595

View details for Web of Science ID 000229436800016

View details for PubMedID 15930630
[Progress and maturation of protein crystallographic experiments]. Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme Wakatsuki, S., Kato, R., Igarashi, N., Kawasaki, M., Matsugaki, N., Hiraki, M., Yamada, Y. 2005; 50 (7): 846-852
View details for PubMedID 15957408
Eap45 in mammalian ESCRT-II binds ubiquitin via a phosphoinositide-interacting GLUE domain JOURNAL OF BIOLOGICAL CHEMISTRY Slagsvold, T., Aasland, R., Hirano, S., Bache, K. G., Raiborg, C., Trambaiolo, D., Wakatsuki, S., Stenmark, H. 2005; 280 (20): 19600-19606
Abstract

Ubiquitination serves as a key sorting signal in the lysosomal degradation of endocytosed receptors through the ability of ubiquitinated membrane proteins to be recognized and sorted by ubiquitin-binding proteins along the endocytic route. The ESCRT-II complex in yeast contains one such protein, Vps36, which harbors a ubiquitin-binding NZF domain and is required for vacuolar sorting of ubiquitinated membrane proteins. Surprisingly, the presumptive mammalian ortholog Eap45 lacks the ubiquitin-binding module of Vps36, and it is thus not clear whether mammalian ESCRT-II functions to bind ubiquitinated cargo. In this paper, we provide evidence that Eap45 contains a novel ubiquitin-binding domain, GLUE (GRAM-like ubiquitin-binding in Eap45), which binds ubiquitin with similar affinity and specificity as other ubiquitin-binding domains. The GLUE domain shares similarities in its primary and predicted secondary structures to phosphoinositide-binding GRAM and PH domains. Accordingly, we find that Eap45 binds to a subset of 3-phosphoinositides, suggesting that ubiquitin recognition could be coordinated with phosphoinositide binding. Furthermore, we show that Eap45 colocalizes with ubiquitinated proteins on late endosomes. These results are consistent with a role for Eap45 in endosomal sorting of ubiquitinated cargo.

View details for DOI 10.1074/jbc.M501510200

View details for Web of Science ID 000229113700023

View details for PubMedID 15755741
Molecular mechanisms of acceptor substrate recognition of a human glucuronyltransferase, GlcAT-P, an enzyme critical in the biosynthesis of the carbohydrate epitope HNK-1 SEIKAGAKU Shiba, T., Kakuda, S., Oka, S., Ishiguro, M., Kawasaki, T., Wakatsuki, S., Kato, R. 2005; 77 (2): 153-158
View details for Web of Science ID 000227397900011

View details for PubMedID 15786744
Crystal structure of the human cytosolic sialidase Neu2 - Evidence for the dynamic nature of substrate recognition JOURNAL OF BIOLOGICAL CHEMISTRY Chavas, L. M., Tringali, C., Fusi, P., Venerando, B., Tettamanti, G., Kato, R., Monti, E., Wakatsuki, S. 2005; 280 (1): 469-475
Abstract

Gangliosides play key roles in cell differentiation, cell-cell interactions, and transmembrane signaling. Sialidases hydrolyze sialic acids to produce asialo compounds, which is the first step of degradation processes of glycoproteins and gangliosides. Sialidase involvement has been implicated in some lysosomal storage disorders such as sialidosis and galactosialidosis. Neu2 is a recently identified human cytosolic sialidase. Here we report the first high resolution x-ray structures of mammalian sialidase, human Neu2, in its apo form and in complex with an inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA). The structure shows the canonical six-blade beta-propeller observed in viral and bacterial sialidases with its active site in a shallow crevice. In the complex structure, the inhibitor lies in the catalytic crevice surrounded by ten amino acids. In particular, the arginine triad, conserved among sialidases, aids in the proper positioning of the carboxylate group of DANA within the active site region. The tyrosine residue, Tyr(334), conserved among mammalian and bacterial sialidases as well as in viral neuraminidases, facilitates the enzymatic reaction by stabilizing a putative carbonium ion in the transition state. The loops containing Glu(111) and the catalytic aspartate Asp(46) are disordered in the apo form but upon binding of DANA become ordered to adopt two short alpha-helices to cover the inhibitor, illustrating the dynamic nature of substrate recognition. The N-acetyl and glycerol moieties of DANA are recognized by Neu2 residues not shared by bacterial sialidases and viral neuraminidases, which can be regarded as a key structural difference for potential drug design against bacteria, influenza, and other viruses.

View details for DOI 10.1074/jbc.M411506200

View details for Web of Science ID 000226025100056

View details for PubMedID 15501818
[The Photon Factory, a synchrotron radiation facility]. Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme Wakatsuki, S., Hiraki, M., Gaponov, Y., Suzuki, M., Igarashi, N., Matsugaki, N. 2004; 49 (11): 1816-1820
View details for PubMedID 15377022
Insights into the phosphoregulation of beta-secretase sorting signal by the VHS domain of GGA1 TRAFFIC Shiba, T., Kametaka, S., Kawasaki, M., Shibata, M., Waguri, S., Uchiyama, Y., Wakatsuki, S. 2004; 5 (6): 437-448
Abstract

BACE (beta-site amyloid precursor protein cleaving enzyme, beta-secretase) is a type-I membrane protein which functions as an aspartic protease in the production of beta-amyloid peptide, a causative agent of Alzheimer's disease. Its cytoplasmic tail has a characteristic acidic-cluster dileucine motif recognized by the VHS domain of adaptor proteins, GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-interacting). Here we show that BACE is colocalized with GGAs in the trans-Golgi network and peripheral structures, and phosphorylation of a serine residue in the cytoplasmic tail enhances interaction with the VHS domain of GGA1 by about threefold. The X-ray crystal structure of the complex between the GGA1-VHS domain and the BACE C-terminal peptide illustrates a similar recognition mechanism as mannose 6-phosphate receptors except that a glutamine residue closes in to fill the gap created by the shorter BACE peptide. The serine and lysine of the BACE peptide point their side chains towards the solvent. However, phosphorylation of the serine affects the lysine side chain and the peptide backbone, resulting in one additional hydrogen bond and a stronger electrostatic interaction with the VHS domain, hence the reversible increase in affinity.

View details for DOI 10.1111/j.1600-0854.2004.00188.x

View details for Web of Science ID 000221542700006

View details for PubMedID 15117318
Structural basis for acceptor substrate recognition of a human glucuronyltransferase, GlcAT-P, an enzyme critical in the biosynthesis of the carbohydrate epitope HNK-1 JOURNAL OF BIOLOGICAL CHEMISTRY Kakuda, S., Shiba, T., Ishiguro, M., Tagawa, H., Oka, S., Kajihara, Y., Kawasaki, T., Wakatsuki, S., Kato, R. 2004; 279 (21): 22693-22703
Abstract

The HNK-1 carbohydrate epitope is found on many neural cell adhesion molecules. Its structure is characterized by a terminal sulfated glucuronyl acid. The glucuronyltransferases, GlcAT-P and GlcAT-S, are involved in the biosynthesis of the HNK-1 epitope, GlcAT-P as the major enzyme. We overexpressed and purified the recombinant human GlcAT-P from Escherichia coli. Analysis of its enzymatic activity showed that it catalyzed the transfer reaction for N-acetyllactosamine (Galbeta1-4GlcNAc) but not lacto-N-biose (Galbeta1-3GlcNAc) as an acceptor substrate. Subsequently, we determined the first x-ray crystal structures of human GlcAT-P, in the absence and presence of a donor substrate product UDP, catalytic Mn(2+), and an acceptor substrate analogue N-acetyllactosamine (Galbeta1-4GlcNAc) or an asparagine-linked biantennary nonasaccharide. The asymmetric unit contains two independent molecules. Each molecule is an alpha/beta protein with two regions that constitute the donor and acceptor substrate binding sites. The UDP moiety of donor nucleotide sugar is recognized by conserved amino acid residues including a DXD motif (Asp(195)-Asp(196)-Asp(197)). Other conserved amino acid residues interact with the terminal galactose moiety of the acceptor substrate. In addition, Val(320) and Asn(321), which are located on the C-terminal long loop from a neighboring molecule, and Phe(245) contribute to the interaction with GlcNAc moiety. These three residues play a key role in establishing the acceptor substrate specificity.

View details for DOI 10.1074/jbc.M400622200

View details for Web of Science ID 000221417100121

View details for PubMedID 14993226
GAT (GGA and Tom1) domain responsible for ubiquitin binding and ubiquitination JOURNAL OF BIOLOGICAL CHEMISTRY Shiba, Y., Katoh, Y., Shiba, T., Yoshino, K., Takatsu, H., Kobayashi, H., Shin, H. W., Wakatsuki, S., Nakayama, K. 2004; 279 (8): 7105-7111
Abstract

GGAs (Golgi-localizing, gamma-adaptin ear domain homology, ADP-ribosylation factor (ARF)-binding proteins) are a family of monomeric adaptor proteins involved in membrane trafficking from the trans-Golgi network to endosomes. The GAT (GGA and Tom1) domains of GGAs have previously been shown to interact with GTP-bound ARF and to be crucial for membrane recruitment of GGAs. Here we show that the C-terminal subdomain of the GAT domain, which is distinct from the N-terminal GAT subdomain responsible for ARF binding, can bind ubiquitin. The binding is mediated by interactions between residues on one side of the alpha3 helix of the GAT domain and those on the so-called Ile-44 surface patch of ubiquitin. The binding of the GAT domain to ubiquitin can be enhanced by the presence of a GTP-bound form of ARF. Furthermore, GGA itself is ubiquitinated in a manner dependent on the GAT-ubiquitin interaction. These results delineate the molecular basis for the interaction between ubiquitin and GAT and suggest that GGA-mediated trafficking is regulated by the ubiquitin system as endosomal trafficking mediated by other ubiquitin-binding proteins.

View details for DOI 10.1074/jbc.M311702200

View details for Web of Science ID 000188969200109

View details for PubMedID 14660606
Secure UNIX socket based controlling system for high throughput protein crystallography experiments JOURNAL OF SYNCHROTRON RADIATION Gaponov, Y., Igarashi, N., Hiraki, M., Sasajima, K., Matsugaki, N., Suzuki, M., Kosuge, T., Wakatsuki, S. 2004; 11: 17-20
Abstract

A control system for high-throughput protein crystallography experiments has been developed based on a multilevel secure (SSL v2/v3) UNIX socket under the Linux operating system. Main features of protein crystallography experiments (purification, crystallization, loop preparation, data collecting, data processing) are dealt with by the software. All information necessary to perform protein crystallography experiments is stored (except raw X-ray data, that are stored in Network File Server) in a relational database (MySQL). The system consists of several servers and clients. TCP/IP secure UNIX sockets with four predefined behaviors [(a) listening to a request followed by a reply, (b) sending a request and waiting for a reply, (c) listening to a broadcast message, and (d) sending a broadcast message] support communications between all servers and clients allowing one to control experiments, view data, edit experimental conditions and perform data processing remotely. The usage of the interface software is well suited for developing well organized control software with a hierarchical structure of different software units (Gaponov et al., 1998), which will pass and receive different types of information. All communication is divided into two parts: low and top levels. Large and complicated control tasks are split into several smaller ones, which can be processed by control clients independently. For communicating with experimental equipment (beamline optical elements, robots, and specialized experimental equipment etc.), the STARS server, developed at the Photon Factory, is used (Kosuge et al., 2002). The STARS server allows any application with an open socket to be connected with any other clients that control experimental equipment. Majority of the source code is written in C/C++. GUI modules of the system were built mainly using Glade user interface builder for GTK+ and Gnome under Red Hat Linux 7.1 operating system.

View details for Web of Science ID 000188920800005

View details for PubMedID 14646123
Automated micro manipulation system with protein crystal PROCEEDINGS OF THE 2004 INTERNATIONAL SYMPOSIUM ON MICRO-NANOMECHATRONICS AND HUMAN SCIENCE Ohara, K., Ohba, K., Tanikawa, T., Hiraki, M., Wakatsuki, S., Mizukawa, M., Tanie, K. 2004: 301-306
View details for Web of Science ID 000227432400054
Integrated controlling system and unified database for high throughput protein crystallography experiments SYNCHROTRON RADIATION INSTRUMENTATION Gaponov, Y. A., Igarashi, N., Hiraki, M., Sasajima, K., Matsugaki, N., Suzuki, M., Kosuge, T., Wakatsuki, S. 2004; 705: 1213-1216
View details for Web of Science ID 000222089000298
The structures and function of GGAs, the traffic controllers at the TGN sorting crossroads CELL STRUCTURE AND FUNCTION Nakayama, K., Wakatsuki, S. 2003; 28 (5): 431-442
Abstract

GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding proteins) are a family of monomeric clathrin adaptor proteins that are conserved from yeasts to humans. Data published during the past four years have provided detailed pictures of the localization, domain organization and structure-function relationships of GGAs. GGAs possess four conserved functional domains, each of which interacts with cargo proteins including mannose 6-phosphate receptors, the small GTPase ARF, clathrin, or accessory proteins including Rabaptin-5 and gamma-synergin. Together with or independent of the adaptor protein complex AP-1, GGAs regulate selective transport of cargo proteins, such as mannose 6-phosphate receptors, from the trans-Golgi network to endosomes mediated by clathrin-coated vesicles.

View details for Web of Science ID 000188824500004

View details for PubMedID 14745135
Molecular mechanism of membrane recruitment of GGA by ARF in lysosomal protein transport NATURE STRUCTURAL BIOLOGY Shiba, T., Kawasaki, M., Takatsu, H., Nogi, T., Matsugaki, N., Igarashi, N., Suzuki, M., Kato, R., Nakayama, K., Wakatsuki, S. 2003; 10 (5): 386-393
Abstract

GGAs are critical for trafficking soluble proteins from the trans-Golgi network (TGN) to endosomes/lysosomes through interactions with TGN-sorting receptors, ADP-ribosylation factor (ARF) and clathrin. ARF-GTP bound to TGN membranes recruits its effector GGA by binding to the GAT domain, thus facilitating recognition of GGA for cargo-loaded receptors. Here we report the X-ray crystal structures of the human GGA1-GAT domain and the complex between ARF1-GTP and the N-terminal region of the GAT domain. When unbound, the GAT domain forms an elongated bundle of three a-helices with a hydrophobic core. Structurally, this domain, combined with the preceding VHS domain, resembles CALM, an AP180 homolog involved in endocytosis. In the complex with ARF1-GTP, a helix-loop-helix of the N-terminal part of GGA1-GAT interacts with the switches 1 and 2 of ARF1 predominantly in a hydrophobic manner. These data reveal a molecular mechanism underlying membrane recruitment of adaptor proteins by ARF-GTP.

View details for DOI 10.1038/nsb920

View details for Web of Science ID 000182536300015

View details for PubMedID 12679809
Structural basis for the accessory protein recruitment by the gamma-adaptin ear domain NATURE STRUCTURAL BIOLOGY Nogi, T., Shiba, Y., Kawasaki, M., Shiba, T., Matsugaki, N., Igarashi, N., Suzuki, M., Kato, R., Takatsu, H., Nakayama, K., Wakatsuki, S. 2002; 9 (7): 527-531
Abstract

The adaptor proteins AP-1 and GGA regulate membrane traffic between the trans-Golgi network (TGN) and endosomes/lysosomes through ARF-regulated membrane association, recognition of sorting signals, and recruitment of clathrin and accessory proteins. The gamma 1-adaptin subunits of AP-1 and GGA possess homologous ear domains involved in the recruitment of accessory proteins, gamma-synergin and Rabaptin-5. The crystal structure of the human gamma 1-adaptin ear domain consists solely of an immunoglobulin-like fold, unlike the alpha-adaptin ear domain. Structure-based mutational analyses reveal a binding site for the accessory proteins that is composed of conserved basic residues, indicating that the recruitment mechanism in gamma 1-adaptin and GGA is distinct from that in alpha-adaptin.

View details for DOI 10.1038/nsb808

View details for Web of Science ID 000176441000012

View details for PubMedID 12042876
Structural basis for recognition of acidic-cluster dileucine sequence by GGA1 NATURE Shiba, T., Takatsu, H., Nogi, T., Matsugaki, N., Kawasaki, M., Igarashi, N., Suzuki, M., Kato, R., Earnest, T., Nakayama, K., Wakatsuki, S. 2002; 415 (6874): 937-941
Abstract

GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-interacting proteins) are critical for the transport of soluble proteins from the trans-Golgi network (TGN) to endosomes/lysosomes by means of interactions with TGN-sorting receptors, ADP-ribosylation factor (ARF), and clathrin. The amino-terminal VHS domains of GGAs form complexes with the cytoplasmic domains of sorting receptors by recognizing acidic-cluster dileucine (ACLL) sequences. Here we report the X-ray structure of the GGA1 VHS domain alone, and in complex with the carboxy-terminal peptide of cation-independent mannose 6-phosphate receptor containing an ACLL sequence. The VHS domain forms a super helix with eight alpha-helices, similar to the VHS domains of TOM1 and Hrs. Unidirectional movements of helices alpha6 and alpha8, and some of their side chains, create a set of electrostatic and hydrophobic interactions for correct recognition of the ACLL peptide. This recognition mechanism provides the basis for regulation of protein transport from the TGN to endosomes/lysosomes, which is shared by sortilin and low-density lipoprotein receptor-related protein.

View details for Web of Science ID 000173941000052

View details for PubMedID 11859376
Purification, crystallization and preliminary X-ray diffraction analysis of the yeast Sec12 Delta p protein, a guanine nucleotide-exchange factor involved in vesicle transport ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Dumon-Seignovert, L., Matsumuto, T., Monaco-Malbet, S., Tomizaki, T., Sato, M., Sato, K., Nakano, A., Wakatsuki, S. 2001; 57: 893-895
Abstract

Sec12 is a guanine nucleotide-exchange factor (GEF) of the GTP-binding protein Sar1. Its GEF activity on Sar1 makes it a key element in vesicle budding from the endoplasmic reticulum to the Golgi apparatus in yeast. Sec12 is an integral membrane glycoprotein of 70 kDa. A 38.5 kDa N-cytoplasmic domain (Sec12Deltap) has been expressed in Saccharomyces cerevisiae and in Escherichia coli, purified to homogeneity and crystallized. Two crystal forms were obtained. Crystal form I belongs to space group P6(2)/P6(4), with unit-cell parameters a = b = 191.7, c = 53.3 A, gamma = 120 degrees, and diffracts to 2.6 A resolution. Crystal form II belongs to space group P1, with unit-cell parameters a = 52.6, b = 53.0, c = 116.8 A, alpha = 98.0, beta = 97.4, gamma = 93.4 degrees, and diffracts to 2.0 A resolution.

View details for Web of Science ID 000169533300027

View details for PubMedID 11375521
Cross-talk and ammonia channeling between active centers in the unexpected domain arrangement of glutamate synthase STRUCTURE Binda, C., Bossi, R. T., Wakatsuki, S., Arzt, S., Coda, A., Curti, B., Vanoni, M. A., Mattevi, A. 2000; 8 (12): 1299-1308
Abstract

The complex iron-sulfur flavoprotein glutamate synthase catalyses the reductive synthesis of L-glutamate from 2-oxoglutarate and L-glutamine, a reaction in the plant and bacterial pathway for ammonia assimilation. The enzyme functions through three distinct active centers carrying out L-glutamine hydrolysis, conversion of 2-oxoglutarate into L-glutamate, and electron uptake from an electron donor.The 3.0 A crystal structure of the dimeric 324 kDa core protein of a bacterial glutamate synthase was solved by the MAD method, using the very weak anomalous signal of the two 3Fe-4S clusters present in the asymmetric unit. The 1,472 amino acids of the monomer fold into a four-domain architecture. The two catalytic domains have canonical Ntn-amidotransferase and FMN binding (beta/alpha)8 barrel folds, respectively. The other two domains have an unusual "cut (beta/alpha)8 barrel" topology and an unexpected novel beta-helix structure. Channeling of the ammonia intermediate is brought about by an internal tunnel of 31 A length, which runs from the site of L-glutamine hydrolysis to the site of L-glutamate synthesis.The outstanding property of glutamate synthase is the ability to coordinate the activity of its various functional sites to avoid wasteful consumption of L-glutamine. The structure reveals two polypeptide segments that connect the catalytic centers and embed the ammonia tunnel, thus being ideally suited to function in interdomain signaling. Depending on the enzyme redox and ligation states, these signal-transducing elements may affect the active site geometry and control ammonia diffusion through a gating mechanism.

View details for Web of Science ID 000168022400011

View details for PubMedID 11188694
Crystal structure of 1-aminocyclopropane-1-carboxylate deaminase from Hartsenula saturnus JOURNAL OF BIOLOGICAL CHEMISTRY Yao, M., Ose, T., Sugimoto, H., Horiuchi, A., Nakagawa, A., Wakatsuki, S., Yokoi, D., Murakami, T., HONMA, M., Tanaka, I. 2000; 275 (44): 34557-34565
Abstract

The pyridoxal 5'-phosphate (PLP)-dependent enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACCD) catalyzes a reaction that involves a ring opening of cyclopropanoid amino acid, yielding alpha-ketobutyrate and ammonia. Unlike other PLP-dependent enzymes, this enzyme has no alpha-hydrogen atom in the substrate. Thus, a unique mechanism for the bond cleavage is expected. The crystal structure of ACCD from Hansenula saturnus has been determined at 2.0 A resolution by the multiple wavelength anomalous diffraction method using mercury atoms as anomalous scatterers. The model was built on the electron density map, which was obtained by the density averaging of multiple crystal forms. The final model was refined to an R-factor of 22.5% and an R(free)-factor of 26.8%. The ACCD folds into two domains, each of which has an open twisted alpha/beta structure similar to the beta-subunit of tryptophan synthase. However, in ACCD, unlike in other members of the beta family of PLP-dependent enzymes, PLP is buried deep in the molecule. The structure provides the first view of the catalytic center of the cyclopropane ring opening.

View details for Web of Science ID 000165095300076

View details for PubMedID 10938279
Structure of tropinone reductase-II complexed with NADP(+) and pseudotropine at 1.9 angstrom resolution: Implication for stereospecific substrate binding and catalysis BIOCHEMISTRY Yamashita, A., Kato, H., Wakatsuki, S., Tomizaki, T., NAKATSU, T., Nakajima, K., Hashimoto, T., Yamada, Y., Oda, J. 1999; 38 (24): 7630-7637
Abstract

Tropinone reductase-II (TR-II) catalyzes the NADPH-dependent reduction of the carbonyl group of tropinone to a beta-hydroxyl group. The crystal structure of TR-II complexed with NADP+ and pseudotropine (psi-tropine) has been determined at 1.9 A resolution. A seven-residue peptide near the active site, disordered in the unliganded structure, is fixed in the ternary complex by participation of the cofactor and substrate binding. The psi-tropine molecule is bound in an orientation which satisfies the product configuration and the stereochemical arrangement toward the cofactor. The substrate binding site displays a complementarity to the bound substrate (psi-tropine) in its correct orientation. In addition, electrostatic interactions between the substrate and Glu156 seem to specify the binding position and orientation of the substrate. A comparison between the active sites in TR-II and TR-I shows that they provide different van der Waals surfaces and electrostatic features. These differences likely contribute to the correct binding mode of the substrates, which are in opposite orientations in TR-II and TR-I, and to different reaction stereospecificities. The active site structure in the TR-II ternary complex also suggests that the arrangement of the substrate, cofactor, and catalytic residues is stereoelectronically favorable for the reaction.

View details for Web of Science ID 000081016200004

View details for PubMedID 10387002
Matching the crystallographic structure of ribosomal protein S7 to a three-dimensional model of the 16S ribosomal RNA RNA-A PUBLICATION OF THE RNA SOCIETY Tanaka, I., Nakagawa, A., Hosaka, H., Wakatsuki, S., Mueller, F., Brimacombe, R. 1998; 4 (5): 542-550
Abstract

Two recently published but independently derived structures, namely the X-ray crystallographic structure of ribosomal protein S7 and the "binding pocket" for this protein in a three-dimensional model of the 16S rRNA, have been correlated with one another. The known rRNA-protein interactions for S7 include a minimum binding site, a number of footprint sites, and two RNA-protein crosslink sites on the 16S rRNA, all of which form a compact group in the published 16S rRNA model (despite the fact that these interactions were not used as primary modeling constraints in building that model). The amino acids in protein S7 that are involved in the two crosslinks to 16S rRNA have also been determined in previous studies, and here we have used these sites to orient the crystallographic structure of S7 relative to its rRNA binding pocket. Some minor alterations were made to the rRNA model to improve the fit. In the resulting structure, the principal positively charged surface of the protein is in contact with the 16S rRNA, and all of the RNA-protein interaction data are satisfied. The quality of the fit gives added confidence as to the validity of the 16S rRNA model. Protein S7 is furthermore known to be crosslinked both to P site-bound tRNA and to mRNA at positions upstream of the P site codon; the matched S7-16S rRNA structure makes a prediction as to the location of this crosslink site within the protein molecule.

View details for Web of Science ID 000073425400005

View details for PubMedID 9582096
ID14 'Quadriga', a beamline for protein crystallography at the ESRF 6th International Conference on Synchrotron Radiation Instrumentation (SRI'97) Wakatsuki, S., Belrhali, H., Mitchell, E. P., Burmeister, W. P., McSweeney, S. M., Kahn, R., Bourgeois, D., Yao, M., Tomizaki, T., Theveneau, P. WILEY-BLACKWELL. 1998: 215–221
Abstract

The ESRF undulator beamline ID14 'Quadriga' is dedicated to monochromatic macromolecular crystallography. Using two undulators with 23 mm and 42 mm periods and a minimum gap of 16 mm installed on a high-beta section, it will provide high-brilliance X-ray beams at around 13.5 keV, as well as a wide tuneability between 6.8 and 40 keV. Based on the Troika concept, this beamline has four simultaneously operating experimental stations: three side stations, EH1, EH2 and EH3, using thin diamond crystals, and an end station, EH4, with a fast-scan double-crystal monochromator. Station EH3 has a kappa-diffractometer, and an off-line Weissenberg camera with a large 80 x 80 cm active area combined with a 2048 x 2048 CCD detector. During data collection the image plates are placed and removed by a robot located inside the hutch using a cassette system. After data collection the image plates are scanned with an off-line drum scanner. Station EH4 is designed for MAD applications, including Xe K-edge anomalous experiments, and is equipped with a 2048 x 2048 CCD detector on a pseudo 2theta arm. A common graphical user interface and a database will be available to cover all aspects of data collection, including strategy optimization. First results on the performance of the optics elements and initial crystallographic results are presented.

View details for Web of Science ID 000074975200017

View details for PubMedID 15263481
Crystal structure of troponin C in complex with troponin I fragment at 2.3-angstrom resolution PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Vassylyev, D. G., Takeda, S., Wakatsuki, S., Maeda, K., Maeda, Y. 1998; 95 (9): 4847-4852
Abstract

Troponin (Tn), the complex of three subunits (TnC, TnI, and TnT), plays a key role in Ca2+-dependent regulation of muscle contraction. To elucidate the interactions between the Tn subunits and the conformation of TnC in the Tn complex, we have determined the crystal structure of TnC (two Ca2+ bound state) in complex with the N-terminal fragment of TnI (TnI1-47). The structure was solved by the single isomorphous replacement method in combination with multiple wavelength anomalous dispersion data. The refinement converged to a crystallographic R factor of 22.2% (Rfree = 32.6%). The central, connecting alpha-helix observed in the structure of uncomplexed TnC (TnCfree) is unwound at the center (residues Ala-87, Lys-88, Gly-89, Lys-90, and Ser-91) and bent by 90 degrees. As a result, TnC in the complex has a compact globular shape with direct interactions between the N- and C-terminal lobes, in contrast to the elongated dumb-bell shaped molecule of uncomplexed TnC. The 31-residue long TnI1-47 alpha-helix stretches on the surface of TnC and stabilizes its compact conformation by multiple contacts with both TnC lobes. The amphiphilic C-end of the TnI1-47 alpha-helix is bound in the hydrophobic pocket of the TnC C-lobe through 38 van der Waals interactions. The results indicate the major difference between Ca2+ receptors integrated with the other proteins (TnC in Tn) and isolated in the cytosol (calmodulin). The TnC/TnI1-47 structure implies a mechanism of how Tn regulates the muscle contraction and suggests a unique alpha-helical regulatory TnI segment, which binds to the N-lobe of TnC in its Ca2+ bound conformation.

View details for Web of Science ID 000073415700014

View details for PubMedID 9560191
The crystal structure of troponin C in complex with N-terminal fragment of troponin I - The mechanism of how the inhibitor action of troponin I is released by Ca2(+)-binding to troponin C MECHANISMS OF WORK PRODUCTION AND WORK ABSORPTION IN MUSCLE Vassylyev, D. G., Takeda, S., Wakatsuki, S., Maeda, K., Maeda, Y. 1998; 453: 157-167
Abstract

Troponin (Tn), the complex of three subunits (TnC, TnI, and TnT), plays a key role in Ca2+ dependent regulation of muscle contraction. To elucidate the interactions between the Tn subunits and the conformation of TnC in the Tn complex, we have determined the crystal structure of TnC in complex with the N-terminal fragment of TnI (TnI1-47). The structure was solved by single isomorphous replacement method in combination with multiple wavelength anomalous dispersion data. The refinement converged to a crystallographic R-factor of 22.2% (R-free = 32.6%). The central, connecting alpha-helix observed in the structure of uncomplexed TnC (TnCfree) is unwound at the center and bent by 90 degrees. As a result, the TnC in the complex has a compact globular shape with direct interactions between the N- and C-lobes, in contrast to the elongated dumb-bell shaped molecule of uncomplexed TnC. The 31-residue long TnI1-47 alpha-helix stretches on the surface of TnC and stabilizes its compact conformation by multiple contacts with both TnC lobes. The amphiphilic C-terminal end of the TnI1-47 alpha-helix is tightly bound in the hydrophobic pocket of the TnC C-lobe through 38 van der Waals interactions. The results indicate the major difference between integrated (TnC) and isolated (calmodulin) Ca2+ receptors. The TnC/TnI1-47 structure suggests the model for a novel regulatory TnI segment bound to TnC and implies the mechanism of how Tn regulates the muscle contraction.

View details for Web of Science ID 000078390400019

View details for PubMedID 9889826
Diamonds, a multilayer and a sagitally focusing crystal as optical elements on the ID14/Quadriga-3 beamline at ESRF Conference on Crystal amd Multilayer Optics Burmeister, W. P., Bourgeois, D., Kahn, R., Belrhali, H., Mitchell, E. P., McSweeney, S. M., Wakatsuki, S. SPIE - INT SOC OPTICAL ENGINEERING. 1998: 188–196
View details for Web of Science ID 000078392600020
Ribosomal protein S7: a new RNA-binding motif with structural similarities to a DNA architectural factor STRUCTURE Hosaka, H., Nakagawa, A., Tanaka, I., Harada, N., Sano, K., Kimura, M., Yao, M., Wakatsuki, S. 1997; 5 (9): 1199-1208
Abstract

The ribosome is a ribonucleoprotein complex which performs the crucial function of protein biosynthesis. Its role is to decode mRNAs within the cell and to synthesize the corresponding proteins. Ribosomal protein S7 is located at the head of the small (30S) subunit of the ribosome and faces into the decoding centre. S7 is one of the primary 16S rRNA-binding proteins responsible for initiating the assembly of the head of the 30S subunit. In addition, S7 has been shown to be the major protein component to cross-link with tRNA molecules bound at both the aminoacyl-tRNA (A) and peptidyl-tRNA (P) sites of the ribosome. The ribosomal protein S7 clearly plays an important role in ribosome function. It was hoped that an atomic-resolution structure of this protein would aid our understanding of ribosomal mechanisms.The structure of ribosomal protein S7 from Bacillus stearothermophilus has been solved at 2.5 A resolution using multiwavelength anomalous diffraction and selenomethionyl-substituted proteins. The molecule consists of a helical hydrophobic core domain and a beta-ribbon arm extending from the hydrophobic core. The helical core domain is composed of a pair of entangled helix-turn-helix motifs; the fold of the core is similar to that of a DNA architectural factor. Highly conserved basic and aromatic residues are clustered on one face of the S7 molecule and create a 16S rRNA contact surface.The molecular structure of S7, together with the results of previous cross-linking experiments, suggest how this ribosomal protein binds to the 3' major domain of 16S rRNA and mediates the folding of 16S rRNA to create the ribosome decoding centre.

View details for Web of Science ID A1997YA41400008

View details for PubMedID 9331423
Production, crystallization, and preliminary x-ray analysis of rabbit skeletal muscle troponin complex consisting of troponin C and fragment (1-47) of troponin I PROTEIN SCIENCE Saijo, Y., Takeda, S., Scherer, A., Kobayashi, T., Maeda, Y., Taniguchi, H., Yao, M., Wakatsuki, S. 1997; 6 (4): 916-918
Abstract

Troponin is a ternary protein complex consisting of subunits TnC. TnI, and TnT, and plays a key role in calcium regulation of the skeletal and cardiac muscle contraction. In the present study, a partial complex (CI47) was prepared from Escherichia coli-expressed rabbit skeletal muscle TnC and fragment 1-47 of TnI, which is obtained by chemical cleavage of an E. coli-expressed mutant of rabbit skeletal muscle TnI. Within the ternary troponin complex, CI47 is thought to form a core that is resistant to proteolytic digestion, and the interaction within CI47 likely maintains the integrity of the troponin complex. Complex CI47 was crystallized in the presence of sodium citrate. The addition of trehalose improved the diffraction pattern of the crystals substantially. The crystal lattice belongs to the space group P3(1)(2)21, with unit cell dimensions a = b = 48.2 A, c = 162 A. The asymmetric unit presumably contains one CI47 complex. Soaking with p-chloromercuribenzenesulfonate (PCMBS) resulted in loss of isomorphism, but enhanced the quality of the crystals. The crystals diffracted up to 2.3 A resolution, with completeness of 91% and R(merge) = 6.4%. The crystals of PCMBS-derivative should be suitable for X-ray studies using the multiple-wavelength anomalous diffraction technique. This is the first step for elucidating the structure of the full troponin complex.

View details for Web of Science ID A1997WR77900020

View details for PubMedID 9098903
Evaluation of Laue diffraction patterns MACROMOLECULAR CRYSTALLOGRAPHY, PT B Clifton, I. J., Duke, E. M., Wakatsuki, S., Ren, Z. 1997; 277: 448-467
View details for Web of Science ID A1997BJ57S00023

View details for PubMedID 18488319
Production and crystallization of lobster muscle tropomyosin expressed in Sf9 cells FEBS LETTERS MIEGEL, A., Sano, K., Yamamoto, K., Maeda, K., Maeda, Y., Taniguchi, H., Yao, M., Wakatsuki, S. 1996; 394 (2): 201-205
Abstract

A new form of muscle tropomyosin crystal has been obtained, by employing new strategies in protein preparation and crystallization. Non-polymerizable tropomyosin was prepared by removing 11 amino acids at the C-terminus. The truncated tropomyosin was expressed in Sf9 insect cells by use of the baculovirus-based expression system, to obtain highly homogeneous protein preparations. By routinely monitoring homogeneity by mass spectrometry, we found that the homogeneity played a key role in obtaining good crystals. The crystal quality was also dependent on isoforms; the crystals raised from a slow muscle-specific isoform diffracted to a higher resolution, compared with a fast muscle-specific counterpart. For crystallization, a high concentration of organic solvent was used as the precipitant; in the presence of 35% DMSO, tetragonal crystals were formed, which belong to space group P4(3)(1)2(1)2 with cell constants of a=b=105.6 angstrom, c=506.9 angstrom. The crystals gave rise to reflections the intensities of which were characteristically determined by the transform of alpha-helical coiled-coil. Thus in the region of 10-5.5 angstrom resolut along the c*-axis, the reflections were weak. For accurate measurement of these reflection intensities, beam-line ID2 in ESRF Grenoble was advantageous owing to the high brilliance and a low background. There the crystals diffracted to beyond 3.0 A along the c*-axis, whereas along the a*-b*-plane reflections were limited to 6.6 angstrom. Data analysis is under way on a data set from a PtCl4 derivative.

View details for Web of Science ID A1996VL91100020

View details for PubMedID 8843164
THE CRYSTAL-STRUCTURE OF CYCLIN-A STRUCTURE Brown, N. R., Noble, M. E., Endicott, J. A., Garman, E. F., Wakatsuki, S., Mitchell, E., Rasmussen, B., Hunt, T., Johnson, L. N. 1995; 3 (11): 1235-1247
Abstract

Eukaryotic cell cycle progression is regulated by cyclin dependent protein kinases (CDKs) whose activity is regulated by association with cyclins and by reversible phosphorylation. Cyclins also determine the subcellular location and substrate specificity of CDKs. Cyclins exhibit diverse sequences but all share homology over a region of approximately 100 amino acids, termed the cyclin box. From the determination of the structure of cyclin A, together with results from biochemical and genetic analyses, we can identify which parts of the cyclin molecular may contribute to cyclin A structure and function.We have solved the crystal structure, at 2.0 A resolution, of an active recombinant fragment of bovine cyclin A, cyclin A-3, corresponding to residues 171-432 of human cyclin A. The cyclin box has an alpha-helical fold comprising five alpha helices. This fold is repeated in the C-terminal region, although this region shares negligible sequence similarity with the cyclin box.Analysis of residues that are conserved throughout the A, B, and E cyclins identifies two exposed clusters of residues, one of which has recently been shown to be involved in the association with human CDK2. The second cluster may identify another site of cyclin A-protein interaction. Comparison of the structure of the unbound cyclin with the structure of cyclin A complexed with CDK2 reveals that cyclin A does not undergo any significant conformational changes on complex formation. Threading analysis shows that the cyclin-box fold is consistent with the sequences of the transcription factor TFIIB and other functionally related proteins. The structural results indicate a role for the cyclin-box fold both as a template for the cyclin family and as a generalised adaptor molecule in the regulation of transcription.

View details for Web of Science ID A1995TG82000013

View details for PubMedID 8591034
A DOUBLE MULTILAYER MONOCHROMATOR AT AN ESRF UNDULATOR FOR MICROBEAM EXPERIMENTS JOURNAL OF SYNCHROTRON RADIATION Deschamps, P., Engstrom, P., Fiedler, S., Riekel, C., Wakatsuki, S., Hoghoj, P., Ziegler, E. 1995; 2: 124-131
Abstract

A water-cooled double W/Si-multilayer monochromator has been operated at an ESRF low-beta undulator beam. For a fixed distance of the two multilayers the first-order Bragg reflection was at ~8 keV. The peak power density of the beam at the exit of the multilayers was ~1 W mm(-2) and the flux density of the first order after a 10 mum collimator was 4 x 10(5) photons s(-1) mum(-2) mA(-1.) The performance of the beam in microbeam diffraction has been tested on a 20 mum W wire. The observed pseudo-Laue pattern is discussed with respect to the multilayer spectrum.

View details for Web of Science ID A1995RP38000002

View details for PubMedID 16714803
LAUE AND MONOCHROMATIC DIFFRACTION STUDIES ON CATALYSIS IN PHOSPHORYLASE-B CRYSTALS PROTEIN SCIENCE Duke, E. M., Wakatsuki, S., Hadfield, A., Johnson, L. N. 1994; 3 (8): 1178-1196
Abstract

The conversion of substrate, heptenitol, to product, beta-1-C-methyl, alpha-D-glucose-1-phosphate (heptulose-2-P), in crystals of glycogen phosphorylase b has been studied by Laue and monochromatic diffraction methods. The phosphorolysis reaction in the crystal was started following liberation of phosphate from a caged phosphate compound, 3,5-dinitrophenyl phosphate (DNPP). The photolysis of DNPP, stimulated by flashes from a xenon flash lamp, was monitored in the crystal with a diode array spectrophotometer. In the Laue diffraction experiments, data to 2.8 A resolution were collected and the first time shot was obtained at 3 min from the start of reaction, and data collection comprised three 800-ms exposures. Careful data processing of Laue photographs for the large enzyme resulted in electron density maps of almost comparable quality to those produced by monochromatic methods. The difference maps obtained from the Laue measurements showed that very little catalysis had occurred 3 min and 1 h after release of phosphate, and a distinct peak consistent with the position expected for phosphate, in the attacking position was observed. Data collection times with monochromatic crystallographic methods on a home source took 16 h for data to 2.3 A resolution. Sufficient phosphate was released from the caged phosphate in the crystal from 5 flashes with a xenon flashlamp within 1 min for the reaction to go to completion within the time scale of the monochromatic data collection procedures. The heptulose-2-P product complex has been refined and the model agrees with that obtained previously with the major difference that the interchange of an aspartic acid (Asp 283) by an arginine (Arg 569) was not observed at the catalytic site. This change is part of the activation process of glycogen phosphorylase and may not have taken place in the current experiments because the caged compound binds weakly at the inhibitor site, restricting conformational change, and because activators of the enzymic reaction were not present in the crystal. In experiments with monochromatic radiation in which low phosphate concentrations were generated either by fewer photons or by diffusion of known phosphate concentrations, mixtures of substrate and product were observed. It was not possible through crystallographic refinement at 2.3 A resolution to establish the fractional occupancies of the enzyme-substrate and enzyme-product complexes, but the results did indicate that the reaction was proceeding slowly, consistent with approximate calculations for the likely rate of the reaction in the crystal.(ABSTRACT TRUNCATED AT 250 WORDS)

View details for Web of Science ID A1994PE33200004

View details for PubMedID 7987213
BLUE FORM OF BACTERIORHODOPSIN AND ITS ORDER-DISORDER TRANSITION DURING DEHYDRATION BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS Wakatsuki, S., Kimura, Y., Stoeckenius, W., Gillis, N., ELIEZER, D., Hodgson, K. O., Doniach, S. 1994; 1185 (2): 160-166
Abstract

Freshly-prepared blue membranes from Halobacterium halobium, previously reported to be disordered, are shown to have a distinct crystal lattice structure, slightly different from the native form. The lattice of the blue form is disrupted irreversibly when dehydrated. The disorder process was observed using time-resolved small-angle X-ray diffraction and analyzed by radial autocorrelation functions. The diffraction peaks of the in-plane lattice first sharpen and increase due to improved membrane orientation, then the trimer lattice becomes disordered and the unit cell dimension decreases by 1.8 A. In contrast, dehydration of purple membranes does not disorder the lattice, and the unit cell dimension shrinks by only 1.0 A. Comparisons of radial autocorrelation functions for the blue membrane during drying show drastic loss of inter-trimer, long-range correlation while the intra-trimer, short-range correlations remain more or less unchanged. This suggests that the deionized protein trimers can maintain their overall structure during the dehydration, even though the lattice dimension decreases appreciably and the two-dimensional crystallinity is disrupted.

View details for Web of Science ID A1994NK09700003

View details for PubMedID 8167134
NEW SOURCES, NEWER DETECTORS, OLD METHODS - THE FUTURE OF PROTEIN CRYSTALLOGRAPHY AT SYNCHROTRONS NUCLEAR INSTRUMENTS & METHODS IN PHYSICS RESEARCH SECTION B-BEAM INTERACTIONS WITH MATERIALS AND ATOMS Clifton, I. J., Wakatsuki, S., Hajdu, J. 1994; 87 (1-4): 76-81
View details for Web of Science ID A1994NJ59700012
LAUE DIFFRACTION STUDY ON THE STRUCTURE OF CYTOCHROME-C PEROXIDASE COMPOUND-I STRUCTURE Fulop, V., Phizackerley, R. P., Soltis, S. M., Clifton, I. J., Wakatsuki, S., ERMAN, J., Hajdu, J., Edwards, S. L. 1994; 2 (3): 201-208
Abstract

Cytochrome c peroxidase from yeast is a soluble haem-containing protein found in the mitochondrial electron transport chain where it probably protects against toxic peroxides. The aim of this study was to obtain a reliable structure for the doubly oxidized transient intermediate (termed compound I) in the reaction of cytochrome c peroxidase with hydrogen peroxide. This intermediate contains a semistable free radical on Trp191, and an oxyferryl haem group.Compound I was produced in crystals of yeast cytochrome c peroxidase by reacting the crystalline enzyme with hydrogen peroxide in a flow cell. The reaction was monitored by microspectrophotometry and Laue crystallography in separate experiments. A nearly complete conversion to compound I was achieved within two minutes of the addition of hydrogen peroxide, and the concentration of the intermediate remained at similar levels for an additional half an hour. The structure of the intermediate was determined by Laue diffraction. The refined Laue structure for compound I shows clear structural changes at the peroxide-binding site but no significant changes at the radical site. The photographs were processed with a new software package (LEAP), overcoming many of the former problems encountered in extracting structural information from Laue exposures.The geometry of the haem environment in this protein allows structural changes to be extremely small, similar in magnitude to those observed for the Fe2+/Fe3+ transition in cytochrome c. The results suggest that these molecules have evolved to transfer electrons with a minimal need for structural adjustment.

View details for Web of Science ID A1994ND06900006

View details for PubMedID 8069633
TIME-RESOLVED DIFFRACTION STUDIES ON GLYCOGEN PHOSPHORYLASE-B PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY A-MATHEMATICAL PHYSICAL AND ENGINEERING SCIENCES Duke, E. M., Hadfield, A., Walters, S., Wakatsuki, S., BRYAN, R. K., Johnson, L. N. 1992; 340 (1657): 245-261
View details for Web of Science ID A1992JK12300011
SMALL-ANGLE X-RAY-SCATTERING DIFFRACTION SYSTEM FOR STUDIES OF BIOLOGICAL AND OTHER MATERIALS AT THE STANFORD-SYNCHROTRON-RADIATION-LABORATORY REVIEW OF SCIENTIFIC INSTRUMENTS Wakatsuki, S., Hodgson, K. O., ELIEZER, D., Rice, M., Hubbard, S., Gillis, N., Doniach, S., SPANN, U. 1992; 63 (2): 1736-1740
View details for Web of Science ID A1992HC46500014
COMPOSITION MODULATION IN AMORPHOUS METAL GERMANIUM ALLOYS DETECTED BY ANOMALOUS SMALL-ANGLE X-RAY-SCATTERING 8TH INTERNATIONAL CONF ON SMALL ANGLE SCATTERING Rice, M., Wakatsuki, S., Bienenstock, A. WILEY-BLACKWELL PUBLISHING, INC. 1991: 598–602
View details for Web of Science ID A1991GL62900026
ANOMALOUS SMALL-ANGLE X-RAY-SCATTERING STUDIES OF METAL-GERMANIUM ALLOYS SYMP ON THIN FILM STRUCTURES AND PHASE STABILITY Rice, M. B., Wakatsuki, S., Bienenstock, A. MATERIALS RESEARCH SOC. 1990: 53–58
View details for Web of Science ID A1990BT05L00008

Professor of Photon Science and of Structural Biology

Photon Science Directorate

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