Molecular mechanism for the substrate specificity of Arthrobacter globiformis M6 α-glucosidase CmmB, belonging to glycoside hydrolase family 13 subfamily 30
Wataru Saburi, Takayoshi Tagami, Takuya Usui, Jian Yu, Toyoyuki Ose, Min Yao, Haruhide Mori
Food Bioscience, 61, 104516, 104516, Elsevier BV, Oct. 2024
Scientific journal

Glycan-shielded homodimer structure and dynamical features of the canine distemper virus hemagglutinin relevant for viral entry and efficient vaccination.
Hideo Fukuhara, Kohei Yumoto, Miyuki Sako, Mizuho Kajikawa, Toyoyuki Ose, Mihiro Kawamura, Mei Yoda, Surui Chen, Yuri Ito, Shin Takeda, Mwila Mwaba, Jiaqi Wang, Takao Hashiguchi, Jun Kamishikiryo, Nobuo Maita, Chihiro Kitatsuji, Makoto Takeda, Kimiko Kuroki, Katsumi Maenaka
eLife, 12, 24 Jul. 2024, [International Magazine]
English, Scientific journal, Canine distemper virus (CDV) belongs to morbillivirus, including measles virus (MeV) and rinderpest virus, which causes serious immunological and neurological disorders in carnivores, including dogs and rhesus monkeys, as recently reported, but their vaccines are highly effective. The attachment glycoprotein hemagglutinin (CDV-H) at the CDV surface utilizes signaling lymphocyte activation molecule (SLAM) and Nectin-4 (also called poliovirus-receptor-like-4; PVRL4) as entry receptors. Although fusion models have been proposed, the molecular mechanism of morbillivirus fusion entry is poorly understood. Here, we determined the crystal structure of the globular head domain of CDV-H vaccine strain at 3.2 Å resolution, revealing that CDV-H exhibits a highly tilted homodimeric form with a six-bladed β-propeller fold. While the predicted Nectin-4-binding site is well conserved with that of MeV-H, that of SLAM is similar but partially different, which is expected to contribute to host specificity. Five N-linked sugars covered a broad area of the CDV-H surface to expose receptor-binding sites only, supporting the effective production of neutralizing antibodies. These features are common to MeV-H, although the glycosylation sites are completely different. Furthermore, real-time observation using high-speed atomic force microscopy revealed highly mobile features of the CDV-H dimeric head via the connector region. These results suggest that sugar-shielded tilted homodimeric structure and dynamic conformational changes are common characteristics of morbilliviruses and ensure effective fusion entry and vaccination.

STAP-2–Derived Peptide Suppresses TCR-Mediated Signals to Initiate Immune Responses
Yuto Sasaki, Kodai Saitoh, Kota Kagohashi, Toyoyuki Ose, Shoya Kawahara, Yuichi Kitai, Ryuta Muromoto, Yuichi Sekine, Michiko Ichii, Akihiko Yoshimura, Kenji Oritani, Jun-ichi Kashiwakura, Tadashi Matsuda
The Journal of Immunology, The American Association of Immunologists, 07 Jul. 2023
Scientific journal, Abstract

Signal-transducing adaptor protein-2 (STAP-2) is an adaptor protein that contains pleckstrin and Src homology 2–like domains, as well as a proline-rich region in its C-terminal region. Our previous study demonstrated that STAP-2 positively regulates TCR signaling by associating with TCR-proximal CD3ζ ITAMs and the lymphocyte-specific protein tyrosine kinase. In this study, we identify the STAP-2 interacting regions of CD3ζ ITAMs and show that the STAP-2–derived synthetic peptide (iSP2) directly interacts with the ITAM sequence and blocks the interactions between STAP-2 and CD3ζ ITAMs. Cell-penetrating iSP2 was delivered into human and murine T cells. iSP2 suppressed cell proliferation and TCR-induced IL-2 production. Importantly, iSP2 treatment suppressed TCR-mediated activation of naive CD4+ T cells and decreased immune responses in CD4+ T cell–mediated experimental autoimmune encephalomyelitis. It is likely that iSP2 is a novel immunomodulatory tool that modulates STAP-2-mediated activation of TCR signaling and represses the progression of autoimmune diseases.

Structural insights into the substrate specificity and activity of a novel mannose 2-epimerase from Runella slithyformis.
Hang Wang, Xiaomei Sun, Wataru Saburi, Saki Hashiguchi, Jian Yu, Toyoyuki Ose, Haruhide Mori, Min Yao
Acta crystallographica. Section D, Structural biology, 79, Pt 7, 585, 595, 01 Jul. 2023, [International Magazine]
English, Scientific journal, Mannose 2-epimerase (ME), a member of the acylglucosamine 2-epimerase (AGE) superfamily that catalyzes epimerization of D-mannose and D-glucose, has recently been characterized to have potential for D-mannose production. However, the substrate-recognition and catalytic mechanism of ME remains unknown. In this study, structures of Runella slithyformis ME (RsME) and its D254A mutant [RsME(D254A)] were determined in their apo forms and as intermediate-analog complexes [RsME-D-glucitol and RsME(D254A)-D-glucitol]. RsME possesses the (α/α)6-barrel of the AGE superfamily members but has a unique pocket-covering long loop (loopα7-α8). The RsME-D-glucitol structure showed that loopα7-α8 moves towards D-glucitol and closes the active pocket. Trp251 and Asp254 in loopα7-α8 are only conserved in MEs and interact with D-glucitol. Kinetic analyses of the mutants confirmed the importance of these residues for RsME activity. Moreover, the structures of RsME(D254A) and RsME(D254A)-D-glucitol revealed that Asp254 is vital for binding the ligand in a correct conformation and for active-pocket closure. Docking calculations and structural comparison with other 2-epimerases show that the longer loopα7-α8 in RsME causes steric hindrance upon binding to disaccharides. A detailed substrate-recognition and catalytic mechanism for monosaccharide-specific epimerization in RsME has been proposed.

Quick and Spontaneous Transformation between [3Fe–4S] and [4Fe–4S] Iron–Sulfur Clusters in the tRNA-Thiolation Enzyme TtuA
Masato Ishizaka, Minghao Chen, Shun Narai, Yoshikazu Tanaka, Toyoyuki Ose, Masaki Horitani, Min Yao
International Journal of Molecular Sciences, 24, 1, 833, 833, MDPI AG, 03 Jan. 2023
Scientific journal, Iron–sulfur (Fe–S) clusters are essential cofactors for enzyme activity. These Fe–S clusters are present in structurally diverse forms, including [4Fe–4S] and [3Fe–4S]. Type-identification of the Fe–S cluster is indispensable in understanding the catalytic mechanism of enzymes. However, identifying [4Fe–4S] and [3Fe–4S] clusters in particular is challenging because of their rapid transformation in response to oxidation–reduction events. In this study, we focused on the relationship between the Fe–S cluster type and the catalytic activity of a tRNA-thiolation enzyme (TtuA). We reconstituted [4Fe–4S]-TtuA, prepared [3Fe–4S]-TtuA by oxidizing [4Fe–4S]-TtuA under strictly anaerobic conditions, and then observed changes in the Fe–S clusters in the samples and the enzymatic activity in the time-course experiments. Electron paramagnetic resonance analysis revealed that [3Fe–4S]-TtuA spontaneously transforms into [4Fe–4S]-TtuA in minutes to one hour without an additional free Fe source in the solution. Although the TtuA immediately after oxidation of [4Fe–4S]-TtuA was inactive [3Fe–4S]-TtuA, its activity recovered to a significant level compared to [4Fe–4S]-TtuA after one hour, corresponding to an increase of [4Fe–4S]-TtuA in the solution. Our findings reveal that [3Fe–4S]-TtuA is highly inactive and unstable. Moreover, time-course analysis of structural changes and activity under strictly anaerobic conditions further unraveled the Fe–S cluster type used by the tRNA-thiolation enzyme.

Structural and molecular insight into antibody recognition of dynamic neoepitopes in membrane tethered MUC1 of pancreatic cancer cells and secreted exosomes
Hajime Wakui, Yasuhiro Yokoi, Chieko Horidome, Toyoyuki Ose, Min Yao, Yoshikazu Tanaka, Hiroshi Hinou, Shin-Ichiro Nishimura
RSC Chemical Biology, Royal Society of Chemistry (RSC), 2023
Scientific journal, We unveil for the first time that pancreatic cancer cells (PANC-1) and secreted exosomes express MUC1 bearing cancer-relevant dynamic epitopes recognized specifically by an anti-MUC1 antibody (SN-131).

A peptide derived from adaptor protein STAP-2 inhibits tumor progression by downregulating epidermal growth factor receptor signaling
Taiga Maemoto, Yuichi Kitai, Haruka Shoji, Runa Takahashi, Shunsuke Yamada, Shiho Takei, Daiki Ito, Ryuta Muromoto, Jun-ichi Kashiwakura, Haruka Handa, Ari Hashimoto, Shigeru Hashimoto, Toyoyuki Ose, Kenji Oritani, Tadashi Matsuda
Journal of Biological Chemistry, in press, 299, 1, 102724, 102724, Dec. 2022, [International Magazine]
English, Scientific journal, Signal-transducing adaptor family member-2 (STAP-2) is an adaptor protein that regulates various intracellular signals. We previously demonstrated that STAP-2 binds to epidermal growth factor receptor (EGFR) and facilitates its stability and activation of EGFR signaling in prostate cancer cells. Inhibition of this interaction may be a promising direction for cancer treatment. Here, we found that 2D5 peptide, a STAP-2-derived peptide, blocked STAP-2-EGFR interactions and suppressed EGFR-mediated proliferation in several cancer cell lines. 2D5 peptide inhibited tumor growth of human prostate cancer cell line DU145 and human lung cancer cell line A549 in murine xenograft models. Additionally, we determined that EGFR signaling and its stability were decreased by 2D5 peptide treatment during EGF stimulation. In conclusion, our study shows that 2D5 peptide is a novel anti-cancer peptide that inhibits STAP-2-mediated activation of EGFR signaling and suppresses prostate and lung cancer progression.

Importance of accessibility to the extracellular juxtamembrane stalk region of membrane protein for substrate recognition by viral ubiquitin ligase K5.
Mizuho Kajikawa, Mizuki Hata, Maho Ishimura, Nanae Imaizumi, Minako Kimura, Kei Miyano, Toyoyuki Ose, Daisuke Asai, Satoshi Ishido, Taisei Kanamoto
The Biochemical journal, 479, 20, 2261, 2278, 28 Oct. 2022, [International Magazine]
English, Scientific journal, Kaposi's sarcoma-associated herpesvirus (KSHV) is a carcinogenic virus that latently infects B cells and causes malignant tumors in immunocompromised patients. KSHV utilizes two viral E3 ubiquitin ligases, K3 and K5, in KSHV-infected cells to mediate the polyubiquitination-dependent down-regulation of several host membrane proteins involved in the immune system. Although K3 and K5 are members of the same family and have similar structural topologies, K3 and K5 have different substrate specificities. Hence, K5 may have a different substrate recognition mode than K3; however, the molecular basis of substrate recognition remains unclear. Here, we investigated the reason why human CD8α, which is known not to be a substrate for both K3 and K5, is not recognized by them, to obtain an understanding for molecular basis of substrate specificity. CD8α forms a disulfide-linked homodimer under experimental conditions to evaluate the viral ligase-mediated down-regulation. It is known that two interchain disulfide linkages in the stalk region between each CD8α monomer (Cys164-Cys164 and Cys181-Cys181) mediate homodimerization. When the interchain disulfide linkage of Cys181-Cys181 was eliminated, CD8α was down-regulated by K5 with a functional RING variant (RINGv) domain via polyubiquitination at the cytoplasmic tail. Aspartic acid, located at the stalk/transmembrane interface of CD8α, was essential for K5-mediated down-regulation of the CD8α mutant without a Cys181-Cys181 linkage. These results suggest that disulfide linkage near the stalk/transmembrane interface critically inhibits substrate targeting by K5. Accessibility to the extracellular juxtamembrane stalk region of membrane proteins may be important for substrate recognition by the viral ubiquitin ligase K5.

Characterization of Single-Chain Fv Fragments of Neutralizing Antibodies to Rabies Virus Glycoprotein.
Kohei Yumoto, Tomoaki Arisaka, Kazuma Okada, Kyosuke Aoki, Toyoyuki Ose, Tatsunori Masatani, Makoto Sugiyama, Naoto Ito, Hideo Fukuhara, Katsumi Maenaka
Viruses, 13, 11, 19 Nov. 2021, [International Magazine]
English, Scientific journal, Rabies has almost a 100% case-fatality rate and kills more than 59,000 people annually around the world. There is no established treatment for rabies. The rabies virus (RABV) expresses only the glycoprotein (RABVG) at the viral surface, and it is the target for the neutralizing antibodies. We previously established mouse monoclonal antibodies, 15-13 and 12-22, which showed neutralizing activity against the RABV, targeting the sequential and conformational epitopes on the RABVG, respectively. However, the molecular basis for the neutralizing activity of these antibodies is not yet fully understood. In this study, we evaluated the binding characteristics of the Fab fragments of the 15-13 and 12-22 antibodies. The recombinant RABVG protein, in prefusion form for the binding analysis, was prepared by the silkworm-baculovirus expression system. Biolayer interferometry (BLI) analysis indicated that the 15-13 Fab interacts with the RABVG, with a KD value at the nM level, and that the 12-22 Fab has a weaker binding affinity (KD ~ μM) with the RABVG compared to the 15-13 Fab. Furthermore, we determined the amino acid sequences of both the antibodies and the designed single-chain Fv fragments (scFvs) of the 15-13 and 12-22 antibodies as another potential biopharmaceutical for targeting rabies. The 15-13 and 12-22 scFvs were successfully prepared by the refolding method and were shown to interact with the RABVG at the nM level and the μM level of the KD, respectively. These binding characteristics were similar to that of each Fab. On the other hand, differential scanning fluorometry (DSF) revealed that the thermal stability of these scFvs decreases compared to their Fabs. While the improvement of the stability of scFvs will still be required, these results provide insights into the neutralizing activity and the potential therapeutic use of antibody fragments for RABV infection.

Structural comparison of the C-terminal domain of functionally divergent lyssavirus P proteins.
Aoi Sugiyama, Tomo Nomai, Xinxin Jiang, Miku Minami, Min Yao, Katsumi Maenaka, Naoto Ito, Paul R Gooley, Gregory W Moseley, Toyoyuki Ose
Biochemical and biophysical research communications, 529, 2, 507, 512, 20 Aug. 2020, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
English, Scientific journal, Lyssavirus P protein is a multifunctional protein that interacts with numerous host-cell proteins. The C-terminal domain (CTD) of P is important for inhibition of JAK-STAT signaling enabling the virus to evade host immunity. Several regions on the surface of rabies virus P are reported to interact with host factors. Among them, an extended, discrete hydrophobic patch of P CTD is notable. Although structures of P CTD of two strains of rabies virus, and of mokola virus have been solved, the structure of P CTD for Duvenhage virus, which is functionally divergent from these species for immune evasion function, is not known. Here, we analyze the structures of P CTD of Duvenhage and of a distinct rabies virus strain to gain further insight on the nature and potential function of the hydrophobic surface. Molecular contacts in crystals suggest that the hydrophobic patch is important to intermolecular interactions with other proteins, which differ between the lyssavirus species.

The Measles Virus V Protein Binding Site to STAT2 Overlaps That of IRF9.
Yuma Nagano, Aoi Sugiyama, Madoka Kimoto, Takuya Wakahara, Yasuyo Noguchi, Xinxin Jiang, Shinya Saijo, Nobutaka Shimizu, Nana Yabuno, Min Yao, Paul R Gooley, Gregory W Moseley, Takashi Tadokoro, Katsumi Maenaka, Toyoyuki Ose
Journal of virology, 94, 17, 17 Aug. 2020, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
English, Scientific journal, Measles virus (MeV) is a highly immunotropic and contagious pathogen that can even diminish preexisting antibodies and remains a major cause of childhood morbidity and mortality worldwide despite the availability of effective vaccines. MeV is one of the most extensively studied viruses with respect to the mechanisms of JAK-STAT antagonism. Of the three proteins translated from the MeV P gene, P and V are essential for inactivation of this pathway. However, the lack of data from direct analyses of the underlying interactions means that the detailed molecular mechanism of antagonism remains unresolved. Here, we prepared recombinant MeV V protein, which is responsible for human JAK-STAT antagonism, and a panel of variants, enabling the biophysical characterization of V protein, including direct V/STAT1 and V/STAT2 interaction assays. Unambiguous direct interactions between the host and viral factors, in the absence of other factors such as Jak1 or Tyk2, were observed, and the dissociation constants were quantified for the first time. Our data indicate that interactions between the C-terminal region of V and STAT2 is 1 order of magnitude stronger than that of the N-terminal region of V and STAT1. We also clarified that these interactions are completely independent of each other. Moreover, results of size exclusion chromatography demonstrated that addition of MeV-V displaces STAT2-core, a rigid region of STAT2 lacking the N- and C-terminal domains, from preformed complexes of STAT2-core/IRF-associated domain (IRF9). These results provide a novel model whereby MeV-V can not only inhibit the STAT2/IRF9 interaction but also disrupt preassembled interferon-stimulated gene factor 3.IMPORTANCE To evade host immunity, many pathogenic viruses inactivate host Janus kinase signal transducer and activator of transcription (STAT) signaling pathways using diverse strategies. Measles virus utilizes P and V proteins to counteract this signaling pathway. Data derived largely from cell-based assays have indicated several amino acid residues of P and V proteins as important. However, biophysical properties of V protein or its direct interaction with STAT molecules using purified proteins have not been studied. We have developed novel molecular tools enabling us to identify a novel molecular mechanism for immune evasion whereby V protein disrupts critical immune complexes, providing a clear strategy by which measles virus can suppress interferon-mediated antiviral gene expression.

Efficient preparation of human and mouse CD1d proteins using silkworm baculovirus expression system.
Hiroki Kusaka, Shunsuke Kita, Takashi Tadokoro, Kouki Yoshida, Yoshiyuki Kasai, Harumi Niiyama, Yukari Fujimoto, Shinya Hanashima, Michio Murata, Shigeru Sugiyama, Toyoyuki Ose, Kimiko Kuroki, Katsumi Maenaka
Protein expression and purification, 172, 105631, 105631, Aug. 2020, [Peer-reviewed], [International Magazine]
English, Scientific journal, CD1d is a major histocompatibility complex (MHC) class I-like glycoprotein and binds to glycolipid antigens that are recognized by natural killer T (NKT) cells. To date, our understanding of the structural basis for glycolipid binding and receptor recognition of CD1d is still limited. Here, we established a preparation method for the ectodomain of human and mouse CD1d using a silkworm-baculovirus expression system. The co-expression of human and mouse CD1d and β2-microglobulin (β2m) in the silkworm-baculovirus system was successful, but the yield of human CD1d was low. A construct of human CD1d fused with β2m via a flexible GS linker as a single polypeptide was prepared to improve protein yield. The production of this single-chained complex was higher (50 μg/larva) than that of the co-expression complex. Furthermore, differential scanning calorimetry revealed that the linker made the CD1d complex more stable and homogenous. These results suggest that the silkworm-baculovirus expression system is useful for structural and biophysical studies of CD1d in several aspects including low cost, easy handling, biohazard-free, rapid, and high yielding.

A straightforward approach to antibodies recognising cancer specific glycopeptidic neoepitopes
Hajime Wakui, Yoshikazu Tanaka, Toyoyuki Ose, Isamu Matsumoto, Koji Kato, Yao Min, Taro Tachibana, Masaharu Sato, Kentaro Naruchi, Fayna Garcia Martin, Hiroshi Hinou, Shin Ichiro Nishimura
Chemical Science, 11, 19, 4999, 5006, 21 May 2020, [Peer-reviewed]
English, Scientific journal, © The Royal Society of Chemistry 2020. Aberrantly truncated immatureO-glycosylation in proteins occurs in essentially all types of epithelial cancer cells, which was demonstrated to be a common feature of most adenocarcinomas and strongly associated with cancer proliferation and metastasis. Although extensive efforts have been made toward the development of anticancer antibodies targeting MUC1, one of the most studied mucins having cancer-relevant immatureO-glycans, no anti-MUC1 antibody recognises carbohydrates and the proximal MUC1 peptide region, concurrently. Here we present a general strategy that allows for the creation of antibodies interacting specifically with glycopeptidic neoepitopes by using homogeneous synthetic MUC1 glycopeptides designed for the streamlined process of immunization, antibody screening, three-dimensional structure analysis, epitope mapping and biochemical analysis. The X-ray crystal structure of the anti-MUC1 monoclonal antibody SN-101 complexed with the antigenic glycopeptide provides for the first time evidence that SN-101 recognises specifically the essential epitope by forming multiple hydrogen bonds both with the proximal peptide and GalNAc linked to the threonine residue, concurrently. Remarkably, the structure of the MUC1 glycopeptide in complex with SN-101 is identical to its solution NMR structure, an extended conformation induced by site-specific glycosylation. We demonstrate that this method accelerates dramatically the development of a new class of designated antibodies targeting a variety of “dynamic neoepitopes” elaborated by disease-specificO-glycosylation in the immunodominant mucin domains and mucin-like sequences found in intrinsically disordered regions of many proteins.

Structure of HIV-2 Nef Reveals Features Distinct from HIV-1 Involved in Immune Regulation.
Kengo Hirao, Sophie Andrews, Kimiko Kuroki, Hiroki Kusaka, Takashi Tadokoro, Shunsuke Kita, Toyoyuki Ose, Sarah L Rowland-Jones, Katsumi Maenaka
iScience, 23, 1, 100758, 100758, 24 Jan. 2020, [Peer-reviewed], [International Magazine]
English, The human immunodeficiency virus (HIV) accessory protein Nef plays a major role in establishing and maintaining infection, particularly through immune evasion. Many HIV-2-infected people experience long-term viral control and survival, resembling HIV-1 elite control. HIV-2 Nef has overlapping but also distinct functions from HIV-1 Nef. Here we report the crystal structure of HIV-2 Nef core. The di-leucine sorting motif forms a helix bound to neighboring molecules, and moreover, isothermal titration calorimetry demonstrated that the CD3 endocytosis motif can directly bind to HIV-2 Nef, ensuring AP-2-mediated endocytosis for CD3. The highly conserved C-terminal region forms a α-helix, absent from HIV-1. We further determined the structure of simian immunodeficiency virus (SIV) Nef harboring this region, demonstrating similar C-terminal α-helix, which may contribute to AP-1 binding for MHC-I downregulation. These results provide insights into the distinct pathogenesis of HIV-2 infection.

Biophysical characterization and single-chain Fv construction of a neutralizing antibody to measles virus.
Takashi Tadokoro, Mst Lubna Jahan, Yuri Ito, Maino Tahara, Surui Chen, Atsutoshi Imai, Natsumi Sugimura, Koki Yoshida, Mizuki Saito, Toyoyuki Ose, Takao Hashiguchi, Makoto Takeda, Hideo Fukuhara, Katsumi Maenaka
The FEBS journal, 287, 1, 145, 159, Jan. 2020, [Peer-reviewed], [International Magazine]
English, The measles virus (MV) is a major cause of childhood morbidity and mortality worldwide. We previously established a mouse monoclonal antibody, 2F4, which shows high neutralizing titers against eight different genotypes of MV. However, the molecular basis for the neutralizing activity of the 2F4 antibody remains incompletely understood. Here, we have evaluated the binding characteristics of a Fab fragment of the 2F4 antibody. Using the MV infectious assay, we demonstrated that 2F4 Fab inhibits viral entry via either of two cellular receptors, SLAM and Nectin4. Surface plasmon resonance (SPR) analysis of recombinant proteins indicated that 2F4 Fab interacts with MV hemagglutinin (MV-H) with a KD value at the nm level. Furthermore, we designed a single-chain Fv fragment of 2F4 antibody as another potential biopharmaceutical to target measles. The stable 2F4 scFv was successfully prepared by the refolding method and shown to interact with MV-H at the μm level. Like 2F4 Fab, scFv inhibited receptor binding and viral entry. This indicates that 2F4 mAb uses the receptor-binding site and/or a neighboring region as an epitope with high affinity. These results provide insight into the neutralizing activity and potential therapeutic use of antibody fragments for MV infection.

Structural and Functional Basis for LILRB Immune Checkpoint Receptor Recognition of HLA-G Isoforms.
Kimiko Kuroki, Haruki Matsubara, Ryo Kanda, Naoyuki Miyashita, Mitsunori Shiroishi, Yuko Fukunaga, Jun Kamishikiryo, Atsushi Fukunaga, Hideo Fukuhara, Kaoru Hirose, Joan S Hunt, Yuji Sugita, Shunsuke Kita, Toyoyuki Ose, Katsumi Maenaka
Journal of immunology (Baltimore, Md. : 1950), 203, 12, 3386, 3394, 15 Dec. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, Human leukocyte Ig-like receptors (LILR) LILRB1 and LILRB2 are immune checkpoint receptors that regulate a wide range of physiological responses by binding to diverse ligands, including HLA-G. HLA-G is exclusively expressed in the placenta, some immunoregulatory cells, and tumors and has several unique isoforms. However, the recognition of HLA-G isoforms by LILRs is poorly understood. In this study, we characterized LILR binding to the β2-microglobulin (β2m)-free HLA-G1 isoform, which is synthesized by placental trophoblast cells and tends to dimerize and multimerize. The multimerized β2m-free HLA-G1 dimer lacked detectable affinity for LILRB1, but bound strongly to LILRB2. We also determined the crystal structure of the LILRB1 and HLA-G1 complex, which adopted the typical structure of a classical HLA class I complex. LILRB1 exhibits flexible binding modes with the α3 domain, but maintains tight contacts with β2m, thus accounting for β2m-dependent binding. Notably, both LILRB1 and B2 are oriented at suitable angles to permit efficient signaling upon complex formation with HLA-G1 dimers. These structural and functional features of ligand recognition by LILRs provide novel insights into their important roles in the biological regulations.

Structural Elucidation of Viral Antagonism of Innate Immunity at the STAT1 Interface.
Hossain MA, Larrous F, Rawlinson SM, Zhan J, Sethi A, Ibrahim Y, Aloi M, Lieu KG, Mok YF, Griffin MDW, Ito N, Ose T, Bourhy H, Moseley GW, Gooley PR
Cell reports, 29, 7, 1934, 1945.e8, Nov. 2019, [Peer-reviewed]

Specificity of Morbillivirus Hemagglutinins to Recognize SLAM of Different Species.
Hideo Fukuhara, Yuri Ito, Miyuki Sako, Mizuho Kajikawa, Koki Yoshida, Fumio Seki, Mwila Hilton Mwaba, Takao Hashiguchi, Masa-Aki Higashibata, Toyoyuki Ose, Kimiko Kuroki, Makoto Takeda, Katsumi Maenaka
Viruses, 11, 8, 19 Aug. 2019, [Peer-reviewed], [International Magazine]
English, Measles virus (MV) and canine distemper virus (CDV) are highly contagious and deadly, forming part of the morbillivirus genus. The receptor recognition by morbillivirus hemagglutinin (H) is important for determining tissue tropism and host range. Recent reports largely urge caution as regards to the potential expansion of host specificities of morbilliviruses. Nonetheless, the receptor-binding potential in different species of morbillivirus H proteins is largely unknown. Herein, we show that the CDV-H protein binds to the dog signaling lymphocyte activation molecule (SLAM), but not to the human, tamarin, or mouse SLAM. In contrast, MV-H can bind to human, tamarin and dog SLAM, but not to that of mice. Notably, MV binding to dog SLAM showed a lower affinity and faster kinetics than that of human SLAM, and MV exhibits a similar entry activity in dog SLAM- and human SLAM-expressing Vero cells. The mutagenesis study using a fusion assay, based on the MV-H-SLAM complex structure, revealed differences in tolerance for the receptor specificity between MV-H and CDV-H. These results provide insights into H-SLAM specificity related to potential host expansion.

¹H, ¹⁵N and ¹³C resonance assignments of the C-terminal domain of the P protein of the Nishigahara strain of rabies virus.
Zhan J, Hossain MA, Sethi A, Ose T, Moseley GW, Gooley PR
Biomolecular NMR assignments, 13, 1, 5, 8, Apr. 2019, [Peer-reviewed]

Neutron crystallographic study of heterotrimeric glutamine amidotransferase CAB.
Li L, Adachi M, Yu J, Kato K, Shinoda A, Ostermann A, Schrader TE, Ose T, Yao M
Acta crystallographica. Section F, Structural biology communications, 75, Pt 3, 193, 196, Mar. 2019, [Peer-reviewed]

Crystal structure of the complex between venom toxin and serum inhibitor from Viperidae snake.
Narumi Shioi, Takashi Tadokoro, Seijiro Shioi, Yuki Okabe, Haruki Matsubara, Shunsuke Kita, Toyoyuki Ose, Kimiko Kuroki, Shigeyuki Terada, Katsumi Maenaka
The Journal of biological chemistry, 294, 4, 1250, 1256, 25 Jan. 2019, [Peer-reviewed], [International Magazine]
English, Venomous snakes have endogenous proteins that neutralize the toxicity of their venom components. We previously identified five small serum proteins (SSP-1-SSP-5) from a highly venomous snake belonging to the family Viperidae as inhibitors of various toxins from snake venom. The endogenous inhibitors belong to the prostate secretory protein of 94 amino acids (PSP94) family. SSP-2 interacts with triflin, which is a member of the cysteine-rich secretory protein (CRISP) family that blocks smooth muscle contraction. However, the structural basis for the interaction and the biological roles of these inhibitors are largely unknown. Here, we determined the crystal structure of the SSP-2-triflin complex at 2.3 Å resolution. A concave region centrally located in the N-terminal domain of triflin is fully occupied by the terminal β-strands of SSP-2. SSP-2 does not bind tightly to the C-terminal cysteine-rich domain of triflin; this domain is thought to be responsible for its channel-blocker function. Instead, the cysteine-rich domain is tilted 7.7° upon binding to SSP-2, and the inhibitor appears to sterically hinder triflin binding to calcium channels. These results help explain how an endogenous inhibitor prevents the venomous protein from maintaining homeostasis in the host. Furthermore, this interaction also sheds light on the binding interface between the human homologues PSP94 and CRISP-3, which are up-regulated in prostate and ovarian cancers.

Structure of MHC class I-like MILL2 reveals heparan-sulfate binding and interdomain flexibility.
Mizuho Kajikawa, Toyoyuki Ose, Yuko Fukunaga, Yuki Okabe, Naoki Matsumoto, Kento Yonezawa, Nobutaka Shimizu, Simon Kollnberger, Masanori Kasahara, Katsumi Maenaka
Nature communications, 9, 1, 4330, 4330, 18 Oct. 2018, [Peer-reviewed], [International Magazine]
English, The MILL family, composed of MILL1 and MILL2, is a group of nonclassical MHC class I molecules that occur in some orders of mammals. It has been reported that mouse MILL2 is involved in wound healing; however, the molecular mechanisms remain unknown. Here, we determine the crystal structure of MILL2 at 2.15 Å resolution, revealing an organization similar to classical MHC class I. However, the α1-α2 domains are not tightly fixed on the α3-β2m domains, indicating unusual interdomain flexibility. The groove between the two helices in the α1-α2 domains is too narrow to permit ligand binding. Notably, an unusual basic patch on the α3 domain is involved in the binding to heparan sulfate which is essential for MILL2 interactions with fibroblasts. These findings suggest that MILL2 has a unique structural architecture and physiological role, with binding to heparan sulfate proteoglycans on fibroblasts possibly regulating cellular recruitment in biological events.

A Sialylated Voltage-Dependent Ca2+ Channel Binds Hemagglutinin and Mediates Influenza A Virus Entry into Mammalian Cells.
Yoichiro Fujioka, Shinya Nishide, Toyoyuki Ose, Tadaki Suzuki, Izumi Kato, Hideo Fukuhara, Mari Fujioka, Kosui Horiuchi, Aya O Satoh, Prabha Nepal, Sayaka Kashiwagi, Jing Wang, Mika Horiguchi, Yuko Sato, Sarad Paudel, Asuka Nanbo, Tadaaki Miyazaki, Hideki Hasegawa, Katsumi Maenaka, Yusuke Ohba
Cell host & microbe, 23, 6, 809, 818, 13 Jun. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, Influenza A virus (IAV) infection is initiated by the attachment of the viral glycoprotein hemagglutinin (HA) to sialic acid on the host cell surface. However, the sialic acid-containing receptor crucial for IAV infection has remained unidentified. Here, we show that HA binds to the voltage-dependent Ca2+ channel Cav1.2 to trigger intracellular Ca2+ oscillations and subsequent IAV entry and replication. IAV entry was inhibited by Ca2+ channel blockers (CCBs) or by knockdown of Cav1.2. The CCB diltiazem also inhibited virus replication in vivo. Reintroduction of wild-type but not the glycosylation-deficient mutants of Cav1.2 restored Ca2+ oscillations and virus infection in Cav1.2-depleted cells, demonstrating the significance of Cav1.2 sialylation. Taken together, we identify Cav1.2 as a sialylated host cell surface receptor that binds HA and is critical for IAV entry.

The Role of Heparan Sulfate Proteoglycans as an Attachment Factor for Rabies Virus Entry and Infection.
Sasaki M, Anindita PD, Ito N, Sugiyama M, Carr M, Fukuhara H, Ose T, Maenaka K, Takada A, Hall WW, Orba Y, Sawa H
The Journal of infectious diseases, 217, 11, 1740, 1749, May 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, Rabies virus (RABV) is the causative agent of fatal neurological disease. Cellular attachment is the initial and essential step for viral infections. Although extensive studies have demonstrated that RABV uses various target cell molecules to mediate infection, no specific molecule has been identified as an attachment factor for RABV infection. Here we demonstrate that cellular heparan sulfate (HS) supports RABV adhesion and subsequent entry into target cells. Enzymatic removal of HS reduced cellular susceptibility to RABV infection, and heparin, a highly sulfated form of HS, blocked viral adhesion and infection. The direct binding between RABV glycoprotein and heparin was demonstrated, and this interaction was shown to require HS N- and 6-O-sulfation. We also revealed that basic amino acids in the ectodomain of RABV glycoprotein serve as major determinants for the RABV-HS interaction. Collectively, our study highlights a previously undescribed role of HS as an attachment factor for RABV infection.

The C-terminal helix of ribosomal P stalk recognizes a hydrophobic groove of elongation factor 2 in a novel fashion.
Tanzawa T, Kato K, Girodat D, Ose T, Kumakura Y, Wieden HJ, Uchiumi T, Tanaka I, Yao M
Nucleic acids research, 46, 6, 3232, 3244, Oxford University Press (OUP), Apr. 2018, [Peer-reviewed]
Scientific journal

39. Insight into polyether natural product biosynthesis mechanism from biochemical, biophysical, and structural aspects of epoxide hydrolase family
Sawada Kohei, Minami Atsushi, Kumeta Hiroyui, Saio Tomohide, Matsumaru Takanori, Oikawa Hideaki, Maenaka Katsumi, Ose Toyoyuki
Symposium on the Chemistry of Natural Products, symposium papers, 60, 229-234, Symposium on the Chemistry of Natural Products Steering Committee, 2018
Japanese, 【背景】
ポリエーテル化合物は，海産微細藻由来のブレベトキシン，放線菌由来のモネンシン，ラサロシド，キジマイシンなどに代表されるように様々な生物が多種多様な骨格を持つポリエーテルを生産している。これら天然物は連続したエーテル環を分子骨格に持ち，生理活性において重要な役割を果たしている。例えば，ポリエーテル骨格による金属イオンのキレーション機構が知られており2, 3, エーテル環上の酸素原子が特定の金属イオンをキレートして電荷を包み込み，外側に疎水性領域の炭素骨格を向ける。これによって細胞内外のイオンの透過性が増加し，抗菌活性などを示す（イオノフォア）。ポリエーテル骨格としては，テトラヒドロフラン（THF）やテトラヒドロピラン（THP）のようなエーテル環が数珠玉状（モネンシンなど），もしくは梯子状（ブレベトキシンなど）に連結されたものである。こうしたポリエーテル系天然物の生合成経路，特に多くの不斉点を有するエーテル環の構築機構は，有機化学的にも非常に興味が持たれてきた。ポリエーテル骨格の構築機構は1983年に提唱されたCane-Celmer-Westley (CCW)モデル「ポリエン-ポリエポキシド仮説」（1）において，環化機構が統一的に説明された。このモデルでは，鎖状ポリオレフィン前駆体がエポキシ化され，生成したポリエポキシドが位置選択的なエポキシド開環反応によりポリエーテル骨格が構築される。

Scheme 1. Monensin B biosynthesis pathway catalyzed by MonBI and MonBII oligomer

私達は，Monensin生合成をモデルケースとしてポリエーテル骨格構築機構の解明に取り組んできた。多様性を決定づけるエーテル環の導入は，エポキシド加水分解酵素ホモログである環化酵素が担うことが，最近は広く知られてる。Monensinの場合，その骨格を構築するために3 回の5-exo 環化反応が必要である (Scheme 1)。しかしながら，モネンシン生合成遺伝子クラスター中には、環化酵素と相同性を持つ遺伝子がmonBI, monBII の2つしか存在しないため、この2つの環化酵素がどのように3 回の環化反応を触媒するかを解明することが，複雑なポリエーテル骨格構築メカニズムを一般化することと同義であると考えた。組換え発現させたMonBI，

Structural and thermodynamic analyses reveal critical features of glycopeptide recognition by the human PILR alpha immune cell receptor
Atsushi Furukawa, Kosuke Kakita, Tomoki Yamada, Mikihiro Ishizuka, Jiro Sakamoto, Nanao Hatori, Naoyoshi Maeda, Fumina Ohsaka, Takashi Saitoh, Takao Nomura, Kimiko Kuroki, Hisanori Nambu, Hisashi Arase, Shigeki Matsunaga, Masahiro Anada, Toyoyuki Ose, Shunichi Hashimoto, Katsumi Maenaka
JOURNAL OF BIOLOGICAL CHEMISTRY, 292, 51, 21128, 21136, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Dec. 2017, [Peer-reviewed]
English, Scientific journal, Before entering host cells, herpes simplex virus-1 uses its envelope glycoprotein B to bind paired immunoglobulin-like type 2 receptor alpha (PILR alpha) on immune cells. PILR alpha belongs to the Siglec (sialic acid (SA)-binding immunoglobulin-like lectin)- like family, members of which bind SA. PILR alpha is the only Siglec member to recognize not only the sialylated O-linked sugar T antigen (sTn) but also its attached peptide region. We previously determined the crystal structure of PILR alpha complexed with the sTn-linked glycopeptide of glycoprotein B, revealing the simultaneous recognition of sTn and peptide by the receptor. However, the contribution of each glycopeptide component to PILR alpha binding was largely unclear. Here, we chemically synthesized glycopeptide derivatives and determined the thermodynamic parameters of their interaction with PILR alpha. We show that glycopeptides with different sugar units linking SA and peptides (i.e. "GlcNAc-type" and "deoxy-GlcNAc-type" glycopeptides) have lower affinity and more enthalpy-driven binding than the wild type (i.e. GalNAc-type glycopeptide). The crystal structures of PILR alpha complexed with these glycopeptides highlighted the importance of stereochemical positioning of the O4 atom of the sugar moiety. These results provide insights both for understanding the unique O-glycosylated peptide recognition by the PILR alpha and for the rational design of herpes simplex virus-1 entry inhibitors.

Rapid Screening by Cell-Based Fusion Assay for Identifying Novel Antivirals of Glycoprotein B-Mediated Herpes Simplex Virus Type 1 Infection
Naoyoshi Maeda, Atsushi Furukawa, Kosuke Kakita, Masahiro Anada, Shunichi Hashimoto, Shigeki Matsunaga, Kimiko Kuroki, Toyoyuki Ose, Akihisa Kato, Jun Arii, Yasushi Kawaguchi, Hisashi Arase, Katsumi Maenaka
BIOLOGICAL & PHARMACEUTICAL BULLETIN, 39, 11, 1897, 1902, PHARMACEUTICAL SOC JAPAN, Nov. 2016, [Peer-reviewed]
English, Scientific journal, Herpes simplex virus type 1 (HSV-1) is a causative agent for a variety of diseases. Although antiherpetic drugs such as acyclovir have been developed to inhibit virus replication through interaction with DNA kinases, their continuous administration leads to an increase in the frequency of drug-resistant HSV-1, which is an important clinical issue that requires urgent solution. Recently, we reported that the sialylated O-linked sugar T antigen (sTn) and its attached peptide region (O-glycosylated sTn peptide) derived from the HSV-1 glycoprotein B (gB) protein inhibited HSV-1 infection by specifically targeting paired immunoglobulin-like type 2 receptor alpha (PILR alpha) in vitro. In this study, to further identify novel inhibitors of gB-mediated HSV-1 infection in vitro, we established a cell-based fusion assay for rapid drug screening. Chinese hamster ovary (CHO) cells were transfected with expression plasmids for HSV-1 gB, gD, gH, and gL, and T7 RNA polymerase, and were designated as the effector cells. The CHO-K1 cells stably expressing PILRa were transfected with the expression plasmid for firefly luciferase under the T7 promoter, and were designated as the target cells. The effector and target cells were co-cultured, and luminescence was measured when both cells were successfully fused. Importantly, we found that cell-to-cell fusion was specifically inhibited by O-glycosylated sTn peptide in a dose dependent manner. Our results suggested that this virus-free cell-based fusion assay system could be a useful and promising approach to identify novel inhibitors of gB-mediated HSV-1 infection, and will aid in the development of antiviral therapeutic strategies for HSV-1-associated diseases.

The immunosuppressive effect of domain-deleted dimer of HLA-G2 isoform in collagen-induced arthritis mice
Ami Takahashi, Kimiko Kuroki, Yuki Okabe, Yoshiyuki Kasai, Naoki Matsumoto, Chisato Yamada, Toshiyuki Takai, Toyoyuki Ose, Shigeyuki Kon, Tadashi Matsuda, Katsumi Maenaka
HUMAN IMMUNOLOGY, 77, 9, 754, 759, ELSEVIER SCIENCE INC, Sep. 2016, [Peer-reviewed]
English, Scientific journal, HLA-G is involved in maternal-fetal immune tolerance and is reported to be a natural tolerogenic molecule. Seven-spliced isoforms including dimeric and beta 2m-free forms have been identified. The major isoform, HLA-G1 (and its soluble type HLA-G5), binds to the inhibitory immune receptors, leukocyte immunoglobulin (Ig)-like receptor (LILR) B1 and LILRB2. We previously reported that HLA-G1 also binds to paired Ig-like receptor (PIR)-B, a mouse homolog of LILRBs, and had a significant immunosuppressive effect in collagen-induced arthritis (CIA) mice. Although HLA-G2 and its soluble form HLA-G6 bind specifically to LILRB2, its functional characteristics are largely unknown. In this study, we report the significant immunosuppressive effect of HLA-G2 dimer in CIA mice. Surface plasmon resonance analysis revealed a specific interaction of HLA-G2 with PIR-B. CIA mice were administered HLA-G2 protein subcutaneously once in the left footpad and clinical severity was evaluated in a double-blind study. A single administration of HLA-G2 maintained a suppressive effect for over 1 month. These results suggested that the HLA-G2 protein might be a useful biopharmaceutical for the treatment of rheumatoid arthritis by binding to inhibitory PIR-B. (C) 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Interaction between a Domain of the Negative Regulator of the Ras-ERK Pathway, SPRED1 Protein, and the GTPase-activating Protein-related Domain of Neurofibromin Is Implicated in Legius Syndrome and Neurofibromatosis Type 1
Yasuko Hirata, Hilde Brems, Mayu Suzuki, Mitsuhiro Kanamori, Masahiro Okada, Rimpei Morita, Isabel Llano-Rivas, Toyoyuki Ose, Ludwine Messiaen, Eric Legius, Akihiko Yoshimura
JOURNAL OF BIOLOGICAL CHEMISTRY, 291, 7, 3124, 3134, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Feb. 2016, [Peer-reviewed]
English, Scientific journal, Constitutional heterozygous loss-of-function mutations in the SPRED1 gene cause a phenotype known as Legius syndrome, which consists of symptoms of multiple cafe-au-lait macules, axillary freckling, learning disabilities, and macrocephaly. Legius syndrome resembles a mild neurofibromatosis type 1 (NF1) phenotype. It has been demonstrated that SPRED1 functions as a negative regulator of the Ras-ERK pathway and interacts with neurofibromin, the NF1 gene product. However, the molecular details of this interaction and the effects of the mutations identified in Legius syndrome and NF1 on this interaction have not yet been investigated. In this study, using a yeast two-hybrid system and an immunoprecipitation assay in HEK293 cells, we found that the SPRED1 EVH1 domain interacts with the N-terminal 16 amino acids and the C-terminal 20 amino acids of the GTPase-activating protein (GAP)-related domain (GRD) of neurofibromin, which form two crossing -helix coils outside the GAP domain. These regions have been shown to be dispensable for GAP activity and are not present in p120(GAP). Several mutations in these N- and C-terminal regions of the GRD in NF1 patients and pathogenic missense mutations in the EVH1 domain of SPRED1 in Legius syndrome reduced the binding affinity between the EVH1 domain and the GRD. EVH1 domain mutations with reduced binding to the GRD also disrupted the ERK suppression activity of SPRED1. These data clearly demonstrate that SPRED1 inhibits the Ras-ERK pathway by recruiting neurofibromin to Ras through the EVH1-GRD interaction, and this study also provides molecular basis for the pathogenic mutations of NF1 and Legius syndrome.

Solution structure of an avirulence protein, AVR-Pia, from Magnaporthe oryzae
Toyoyuki Ose, Azusa Oikawa, Yukiko Nakamura, Katsumi Maenaka, Yuya Higuchi, Yuki Satoh, Shiho Fujiwara, Makoto Demura, Teruo Sone, Masakatsu Kamiya
JOURNAL OF BIOMOLECULAR NMR, 63, 2, 229, 235, SPRINGER, Oct. 2015, [Peer-reviewed]
English, Scientific journal

Crystal structure of extracellular domain of human lectin-like transcript 1 (LLT1), the ligand for natural killer receptor-P1A
Shunsuke Kita, Haruki Matsubara, Yoshiyuki Kasai, Takaharu Tamaoki, Yuki Okabe, Hideo Fukuhara, Jun Kamishikiryo, Elena Krayukhina, Susumu Uchiyama, Toyoyuki Ose, Kimiko Kuroki, Katsumi Maenaka
EUROPEAN JOURNAL OF IMMUNOLOGY, 45, 6, 1605, 1613, WILEY-BLACKWELL, Jun. 2015, [Peer-reviewed]
English, Scientific journal, Emerging evidence has revealed the pivotal roles of C-type lectin-like receptors (CTLRs) in the regulation of a wide range of immune responses. Human natural killer cell receptor-P1A (NKRP1A) is one of the CTLRs and recognizes another CTLR, lectin-like transcript 1 (LLT1) on target cells to control NK, NKT and Th17 cells. The structural basis for the NKRP1A-LLT1 interaction was limitedly understood. Here, we report the crystal structure of the ectodomain of LLT1. The plausible receptor-binding face of the C-type lectin-like domain is flat, and forms an extended -sheet. The residues of this face are relatively conserved with another CTLR, keratinocyte-associated C-type lectin, which binds to the CTLR member, NKp65. A LLT1-NKRP1A complex model, prepared using the crystal structures of LLT1 and the keratinocyte-associated C-type lectin-NKp65 complex, reasonably satisfies the charge consistency and the conformational complementarity to explain a previous mutagenesis study. Furthermore, crystal packing and analytical ultracentrifugation revealed dimer formation, which supports a complex model. Our results provide structural insights for understanding the binding modes and signal transduction mechanisms, which are likely to be conserved in the CTLR family, and for further rational drug design towards regulating the LLT1 function.

Roquin binds microRNA-146a and Argonaute2 to regulate microRNA homeostasis
Monika Srivastava, Guowen Duan, Nadia J. Kershaw, Vicki Athanasopoulos, Janet H. C. Yeo, Toyoyuki Ose, Desheng Hu, Simon H. J. Brown, Slobodan Jergic, Hardip R. Patel, Alvin Pratama, Sashika Richards, Anil Verma, E. Yvonne Jones, Vigo Heissmeyer, Thomas Preiss, Nicholas E. Dixon, Mark M. W. Chong, Jeffrey J. Babon, Carola G. Vinuesa
NATURE COMMUNICATIONS, 6, 6253, NATURE PUBLISHING GROUP, Feb. 2015, [Peer-reviewed]
English, Scientific journal, Roquin is an RNA-binding protein that prevents autoimmunity and inflammation via repression of bound target mRNAs such as inducible costimulator (Icos). When Roquin is absent or mutated (Roquin(san)), Icos is overexpressed in T cells. Here we show that Roquin enhances Dicer-mediated processing of pre-miR-146a. Roquin also directly binds Argonaute2, a central component of the RNA-induced silencing complex, and miR-146a, a microRNA that targets Icos mRNA. In the absence of functional Roquin, miR-146a accumulates in T cells. Its accumulation is not due to increased transcription or processing, rather due to enhanced stability of mature miR-146a. This is associated with decreased 3' end uridylation of the miRNA. Crystallographic studies reveal that Roquin contains a unique HEPN domain and identify the structural basis of the 'san' mutation and Roquin's ability to bind multiple RNAs. Roquin emerges as a protein that can bind Ago2, miRNAs and target mRNAs, to control homeostasis of both RNA species.

Structural Characterization Reveals the Keratinolytic Activity of an Arthrobacter nicotinovorans Protease
Teruo Sone, Yumiko Haraguchi, Aki Kuwahara, Toyoyuki Ose, Megumi Takano, Ayumi Abe, Michiko Tanaka, Isao Tanaka, Kozo Asano
PROTEIN AND PEPTIDE LETTERS, 22, 1, 63, 72, BENTHAM SCIENCE PUBL LTD, 2015, [Peer-reviewed]
English, Scientific journal, Elevated cadmium (Cd) concentrations in fishery byproducts are an environmental concern, that might be reduced by enzymatic removal and adsorption of the contaminants during recycling the byproducts as animal food. We cloned the gene for Arthrobacter nicotinovorans serine protease (ANISEP), which was isolated from the hepatopancreas of the Japanese scallop (Patiopecten yessoensis) and has been found to be an effective enzyme for Cd(II) removal. The gene is 993 bp in length and encodes 330 amino acids, including the pre (1-30) and pro (31-111) sequences. The catalytic triad consists of His, Asp, and Ser. Sequence similarities indicate that ANISEP is a extracellular serine protease. X-ray crystallography revealed structural similarities between ANISEP and the trypsin-like serine protease NAALP from Nesterenkonia sp. Site-directed mutagenesis identified Ser171 as catalytic residue. The keratinolytic activity of ANISEP was 10-fold greater than that of trypsin. ANISEP digested Cd(II)-bound recombinant metallothionein MT-10a from Laternula elliptica, but did not release Cd. These results further suggest ANISEP is a trypsin-like serine protease that can release Cd from the Japanese scallop hepatopancreas because of its strong keratinolytic activity.

X-ray structures of eIF5B and the eIF5B-eIF1A complex: the conformational flexibility of eIF5B is restricted on the ribosome by interaction with eIF1A
Aiping Zheng, Jian Yu, Reo Yamamoto, Toyoyuki Ose, Isao Tanaka, Min Yao
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 70, Pt 12, 3090, 3098, WILEY-BLACKWELL, Dec. 2014, [Peer-reviewed]
English, Scientific journal, eIF5B and eIF1A are two translation-initiation factors that are universally conserved among all kingdoms. They show a unique interaction in eukaryotes which is important for ribosomal subunit joining. Here, the structures of two isolated forms of yeast eIF5B and of the eIF5B-eIF1A complex (eIF1A and eIF5B do not contain the respective N-terminal domains) are reported. The eIF5B-eIF1A structure shows that the C-terminal tail of eIF1A binds to eIF5B domain IV, while the core domain of eIF1A is invisible in the electron-density map. Although the individual domains in all structures of eIF5B or archaeal IF5B (aIF5B) are similar, their domain arrangements are significantly different, indicating high structural flexibility, which is advantageous for conformational change during ribosomal subunit joining. Based on these structures, models of eIF5B, eIF1A and tRNA(i)(Met) on the 80S ribosome were built. The models suggest that the interaction between the eIF1A C-terminal tail and eIF5B helps tRNA(i)(Met) to bind to eIF5B domain IV, thus preventing tRNA(i)(Met) dissociation, stabilizing the interface for subunit joining and providing a checkpoint for correct ribosome assembly.

Structural basis for simultaneous recognition of an O-glycan and its attached peptide of mucin family by immune receptor PILR alpha
Kimiko Kuroki, Jing Wang, Toyoyuki Ose, Munechika Yamaguchi, Shigekazu Tabata, Nobuo Maita, Seiko Nakamura, Mizuho Kajikawa, Amane Kogure, Takeshi Satoh, Hisashi Arase, Katsumi Maenaka
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 111, 24, 8877, 8882, NATL ACAD SCIENCES, Jun. 2014, [Peer-reviewed], [Lead author]
English, Scientific journal, Paired Ig-like type 2 receptor a (PILR alpha) recognizes a wide range of O-glycosylated mucin and related proteins to regulate broad immune responses. However, the molecular characteristics of these recognitions are largely unknown. Here we show that sialylated O-linked sugar T antigen (sTn) and its attached peptide region are both required for ligand recognition by PILR alpha. Furthermore, we determined the crystal structures of PILR alpha and its complex with an sTn and its attached peptide region. The structures show that PILR alpha exhibits large conformational change to recognize simultaneously both the sTn O-glycan and the compact peptide structure constrained by proline residues. Binding and functional assays support this binding mode. These findings provide significant insight into the binding motif and molecular mechanism (which is distinct from sugar-recognition receptors) by which O-glycosylated mucin proteins with sTn modifications are recognized in the immune system as well as during viral entry.

Heterologous production, purification, and immunodetection of Magnaporthe oryzae avirulence protein AVR-Pia
Yuki Satoh, Shinsuke Miki, Toyoyuki Ose, Azusa Oikawa, Katsumi Maenaka, Ryouhei Terauchi, Kozo Asano, Teruo Sone
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 78, 4, 680, 686, TAYLOR & FRANCIS LTD, Apr. 2014, [Peer-reviewed]
English, Scientific journal, The avirulence gene AVR-Pia of Magnaporthe oryzae, which induces a hypersensitive reaction in rice cultivars containing the resistance gene Pia, was expressed in Escherichia coli. AVR-Pia protein was collected as inclusion bodies, denatured, and refolded. Finally, recombinant AVR-Pia (rAVR-Pia) protein was purified by column chromatography. Infiltration of rAVR-Pia triggered cell browning in the leaves of rice cultivar Aichiasahi (Pia), with accumulation of H2O2 and induction of PR1a expression in rice. On the other hand, these reactions were not observed in Shin-2 (pia) leaves after the same treatment. This observation indicated that rAVR-Pia had the function of an avirulence protein. rAVR-Pia was used for immunization of a rabbit, and anti-AVR-Pia antiserum was prepared. The specificity of this antibody was appraised by detecting native AVR-Pia in the inoculated leaf sheath extract using Western blotting in combination with immunoprecipitation. Native AVR-Pia was successfully detected, and its molecular weight was estimated to be 7.4 kDa, indicating signal peptide cleavage. Additionally, secreted native AVR-Pia was quantified as 3.7 ng/g rice sheath.

Allosteric Regulation of Epoxide Opening Cascades by a Pair of Epoxide Hydrolases in Monensin Biosynthesis
Atsushi Minami, Toyoyuki Ose, Kyohei Sato, Azusa Oikawa, Kimiko Kuroki, Katsumi Maenaka, Hiroki Oguri, Hideaki Oikawa
ACS CHEMICAL BIOLOGY, 9, 2, 562, 569, AMER CHEMICAL SOC, Feb. 2014, [Peer-reviewed]
English, Scientific journal, Multistep catalysis of epoxide hydrolase/cyclase in the epoxide opening cascade is an intriguing issue in polyether biosynthesis. A pair of structurally homologous epoxide hydrolases was found in gene clusters of ionophore polyethers. In the epoxide opening reactions with MonBI and MonBII involved in monensin biosynthesis, we found that MonBII and catalytically inactive MonBI mutant catalyzed two-step reactions of bisepoxide substrate analogue to afford bicyclic product although MonBII alone catalyzed only the first cyclization. The X-ray crystal structure of MonBI dimers suggested the importance of the KSD motif in MonBI/MonBI interaction, which was further supported by gel filtration chromatography of wild-type MonBI and mutant MonBI. The involvement of the KSD motif in heterodimer formation was confirmed by in vitro assay. Direct evidence of MonBI/MonBII interaction was obtained by native mass spectrometry. Its dissociation constant was determined as 2.21 X 10(-5) M by surface plasmon resonance. Our results suggested the involvement of an allosteric regulation mechanism by MonBI/MonBII interaction in monensin skeletal construction.

NKG2D Triggers Cytotoxicity in Murine Epidermal gamma delta T Cells via PI3K-Dependent, Syk/ZAP70-Independent Signaling Pathway
Atsuko Ibusuki, Kazuhiro Kawai, Shigeru Yoshida, Youhei Uchida, Ayano Nitahara-Takeuchi, Kimiko Kuroki, Mizuho Kajikawa, Toyoyuki Ose, Katsumi Maenaka, Masanori Kasahara, Takuro Kanekura
JOURNAL OF INVESTIGATIVE DERMATOLOGY, 134, 2, 396, 404, NATURE PUBLISHING GROUP, Feb. 2014, [Peer-reviewed]
English, Scientific journal, Murine epidermal gamma delta T cells, known as dendritic epidermal T cells (DETCs), survey tissue stress through the invariant T-cell receptor (TCR) and non-clonotypic receptors such as NKG2D. NKG2D signaling via the DAP10-phosphatidylinositol 3-kinase (PI3K) pathway directly stimulates cytotoxicity in natural killer (NK) cells and costimulates CD8(+) T cells to augment TCR signals. In activated murine NK cells, NKG2D signals also via the DAP12-Syk/ZAP70 pathway that triggers both cytotoxicity and cytokine production. It remains controversial whether NKG2D on DETCs is a primary activating receptor or functions only as a costimulatory receptor, and signaling pathways initiated by NKG2D ligation in DETCs have not been analyzed. We show that stimulation of short-term DETC lines with recombinant NKG2D ligands triggers degranulation (exocytosis of cytotoxic granules) via the PI3K-dependent signaling pathway, but does not induce cytokine production or Syk/ZAP70 activation. Coengagement of TCR or Syk/ZAP70 signaling was not crucial for DETC-mediated killing of NKG2D ligand-expressing target cells. Thus, NKG2D can function as a coactivating stress receptor that directly triggers cytotoxicity in DETCs, at least after priming, via the PI3K-dependent, Syk/ZAP70-independent signaling pathway.

Crystal Structure of the Lamprey Variable Lymphocyte Receptor C Reveals an Unusual Feature in Its N-Terminal Capping Module
Ryo Kanda, Yoichi Sutoh, Jun Kasamatsu, Katsumi Maenaka, Masanori Kasahara, Toyoyuki Ose
PLOS ONE, 9, 1, e85875, PUBLIC LIBRARY SCIENCE, Jan. 2014, [Peer-reviewed]
English, Scientific journal, Jawless vertebrates represented by lampreys and hagfish use variable lymphocyte receptors (VLRs) as antigen receptors to mount adaptive immune responses. VLRs generate diversity that is comparable to immunoglobulins and T-cell receptors by a gene conversion-like mechanism, which is mediated by cytosine deaminases. Currently, three types of VLRs, VLRA, VLRB, and VLRC, have been identified in lampreys. Crystal structures of VLRA and VLRB in complex with antigens have been reported recently, but no structural information is available for VLRC. Here, we present the first crystal structure of VLRC from the Japanese lamprey (Lethenteron japonicum). Similar to VLRA and VLRB, VLRC forms a typical horseshoe-like solenoid structure with a variable concave surface. Strikingly, its N-terminal cap has a long loop with limited sequence variability that protrudes toward the concave surface, which is the putative antigen-binding surface. Furthermore, as predicted previously, its C-terminal cap lacks a highly variable protruding loop that plays an important role in antigen recognition by lamprey VLRA and VLRB. Recent work suggests that VLRC+ lymphocytes in jawless vertebrates might be akin to gamma delta T cells in jawed vertebrates. Structural features of lamprey VLRC described here suggest that it may recognize antigens in a unique manner.

Erratum: Structure analysis of geranyl pyrophosphate methyltransferase and the proposed reaction mechanism of SAM-dependent C-methylation (Acta Crystallographica Section D: Biological Crystallography (2012) (1558-1569)
Orapin Ariyawutthiphan, Toyoyuki Ose, Atsushi Minami, Sandip Shinde, Muneya Tsuda, Yong-Gui Gao, Min Yao, Hideaki Oikawa, Isao Tanaka
Acta Crystallographica Section D: Biological Crystallography, 69, 12, 2584, Dec. 2013, [Peer-reviewed]
English, Scientific journal

Structural analysis for glycolipid recognition by the C-type lectins Mincle and MCL
Atsushi Furukawa, Jun Kamishikiryo, Daiki Mori, Kenji Toyonaga, Yuki Okabe, Aya Toji, Ryo Kanda, Yasunobu Miyake, Toyoyuki Ose, Sho Yamasaki, Katsumi Maenaka
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 110, 43, 17438, 17443, NATL ACAD SCIENCES, Oct. 2013, [Peer-reviewed]
English, Scientific journal, Mincle [macrophage inducible Ca2+-dependent (C-type) lectin; CLEC4E] and MCL (macrophage C-type lectin; CLEC4D) are receptors for the cord factor TDM (trehalose-6,6'-dimycolate), a unique glycolipid of mycobacterial cell-surface components, and activate immune cells to confer adjuvant activity. Although it is known that receptor-TDM interactions require both sugar and lipid moieties of TDM, the mechanisms of glycolipid recognition by Mincle and MCL remain unclear. We here report the crystal structures of Mincle, MCL, and the Mincle-citric acid complex. The structures revealed that these receptors are capable of interacting with sugar in a Ca2+-dependent manner, as observed in other C-type lectins. However, Mincle and MCL uniquely possess shallow hydrophobic regions found adjacent to their putative sugar binding sites, which reasonably locate for recognition of fatty acid moieties of glycolipids. Functional studies using mutant receptors as well as glycolipid ligands support this deduced binding mode. These results give insight into the molecular mechanism of glycolipid recognition through C-type lectin receptors, which may provide clues to rational design for effective adjuvants.

Distinct HIV-1 Escape Patterns Selected by Cytotoxic T Cells with Identical Epitope Specificity
Yuichi Yagita, Nozomi Kuse, Kimiko Kuroki, Hiroyuki Gatanaga, Jonathan M. Carlson, Takayuki Chikata, Zabrina L. Brumme, Hayato Murakoshi, Tomohiro Akahoshi, Nico Pfeifer, Simon Mallal, Mina John, Toyoyuki Ose, Haruki Matsubara, Ryo Kanda, Yuko Fukunaga, Kazutaka Honda, Yuka Kawashima, Yasuo Ariumi, Shinichi Oka, Katsumi Maenaka, Masafumi Takiguchi
JOURNAL OF VIROLOGY, 87, 4, 2253, 2263, AMER SOC MICROBIOLOGY, Feb. 2013, [Peer-reviewed]
English, Scientific journal, Pol283-8-specific, HLA-B*51:01-restricted, cytotoxic T cells (CTLs) play a critical role in the long-term control of HIV-1 infection. However, these CTLs select for the reverse transcriptase (RT) I135X escape mutation, which may be accumulating in circulating HIV-1 sequences. We investigated the selection of the I135X mutation by CTLs specific for the same epitope but restricted by HLA-B*52:01. We found that Pol283-8-specific, HLA-B*52:01-restricted CTLs were elicited predominantly in chronically HIV-1-infected individuals. These CTLs had a strong ability to suppress the replication of wild-type HIV-1, though this ability was weaker than that of HLA-B*51:01-restricted CTLs. The crystal structure of the HLA-B*52:01-Pol283-8 peptide complex provided clear evidence that HLA-B*52:01 presents the peptide similarly to HLA-B*51:01, ensuring the cross-presentation of this epitope by both alleles. Population level analyses revealed a strong association of HLA-B*51:01 with the I135T mutant and a relatively weaker association of HLA-B*52:01 with several I135X mutants in both Japanese and predominantly Caucasian cohorts. An in vitro viral suppression assay revealed that the HLA-B*52:01-restricted CTLs failed to suppress the replication of the I135X mutant viruses, indicating the selection of these mutants by the CTLs. These results suggest that the different pattern of I135X mutant selection may have resulted from the difference between these two CTLs in the ability to suppress HIV-1 replication.

Structure analysis of geranyl pyrophosphate methyltransferase and the proposed reaction mechanism of SAM-dependent C-methylation
Orapin Ariyawutthiphan, Toyoyuki Ose, Atsushi Minami, Sandip Sinde, Muneya Tsuda, Yong-Gui Gao, Min Yao, Hideaki Oikawa, Isao Tanaka
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 68, 1558, 1569, WILEY-BLACKWELL, Nov. 2012, [Peer-reviewed]
English, Scientific journal, In the typical isoprenoid-biosynthesis pathway, condensation of the universal C-5-unit precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) occurs via the common intermediates prenyl pyrophosphates (C-10-C-20). The diversity of isoprenoids reflects differences in chain length, cyclization and further additional modification after cyclization. In contrast, the biosynthesis of 2-methylisonorneol (2-MIB), which is responsible for taste and odour problems in drinking water, is unique in that it primes the enzymatic methylation of geranyl pyrophosphate (GPP) before cyclization, which is catalyzed by an S-adenosyl-L-methionine-dependent methyltransferase (GPPMT). The substrate of GPPMT contains a nonconjugated olefin and the reaction mechanism is expected to be similar to that of the steroid methyltransferase (SMT) family. Here, structural analysis of GPPMT in complex with its cofactor and substrate revealed the mechanisms of substrate recognition and possible enzymatic reaction. Using the structures of these complexes, methyl-group transfer and the subsequent proton-abstraction mechanism are discussed. GPPMT and SMTs contain a conserved glutamate residue that is likely to play a role as a general base. Comparison with the reaction mechanism of the mycolic acid cyclopropane synthase (MACS) family also supports this result. This enzyme represented here is the first model of the enzymatic C-methylation of a nonconjugated olefin in the isoprenoid-biosynthesis pathway. In addition, an elaborate system to avoid methylation of incorrect substrates is proposed.

Simultaneous inhibition of Src and Aurora kinases by SU6656 induces therapeutic synergy in human synovial sarcoma growth, invasion and angiogenesis in vivo
Ryuta Arai, Masumi Tsuda, Takuya Watanabe, Toyoyuki Ose, Chikashi Obuse, Katsumi Maenaka, Akio Minami, Yusuke Ohba
EUROPEAN JOURNAL OF CANCER, 48, 15, 2417, 2430, ELSEVIER SCI LTD, Oct. 2012, [Peer-reviewed]
English, Scientific journal, Synovial sarcoma is an obstinate, high-grade malignancy because of its modest responses to radiotherapy and chemotherapy; the identification of effective therapeutics for this sarcoma is therefore necessary. Inhibition of Src family kinases (SFKs) suppresses the proliferation of synovial sarcoma cells in vitro, as we have previously reported. In this study, to validate the efficacy of Src inhibition in vivo, we employed SU6656, which was originally identified as a specific SFK inhibitor. SU6656 treatment significantly impaired the growth of established, existing tumours formed by synovial sarcoma cells in mice. Tumour cell invasion into the surrounding tissues was also abolished by SU6656. It is noteworthy that SU6656 but not PP2 induced a defect in cleavage furrow formation during cytokinesis, resulting in G2/M accumulation and subsequent apoptosis. Intriguingly, SU6656 abrogated the catalytic activities of Aurora kinases and led to the down-regulation of phosphorylated histone H3 coincidently with p53 accumulation, as did the Aurora kinase inhibitor VX-680. Structural comparison indicated an extensive similarity between the catalytic domains of SFKs and Aurora kinases. The structural analysis also revealed the potential binding mode of SU6656 to the ATP-binding cleft of Aurora B via four hydrogen bonds. SU6656 prevented angiogenesis within the tumours by attenuating vascular endothelial growth factor (VEGF) production by tumour cells and the subsequent chemotaxis of endothelial cells; these effects were the result of the inhibition of SFKs but not Aurora kinases. Based on these results, we hereby report a novel property of SU6656 as a dual inhibitor of SFKs and Aurora kinases, the suppression of both of which effectively abrogates tumour development and the progression of synovial sarcoma in vivo. (C) 2011 Elsevier Ltd. All rights reserved.

Crystallization Strategy for the Glycoprotein-Receptor Complex Between Measles Virus Hemagglutinin and Its Cellular Receptor SLAM
Takao Hashiguchi, Toyoyuki Ose, Marie Kubota, Nobuo Maita, Jun Kamishikiryo, Katsumi Maenaka, Yusuke Yanagi
PROTEIN AND PEPTIDE LETTERS, 19, 4, 468, 473, BENTHAM SCIENCE PUBL LTD, Apr. 2012, [Peer-reviewed]
English, Scientific journal, Measles virus (MV), one of the most contagious agents, infects immune cells using the signaling lymphocyte activation molecule (SLAM) on the cell surface. A complex of SLAM and the attachment protein, hemagglutinin (MV-H), has remained elusive due to the intrinsic handling difficulty including glycosylation. Furthermore, crystals obtained of this complex are either nondiffracting or poorly-diffracting. To solve this problem, we designed a systematic approach using a combination of the following techniques; (1) a transient expression system in HEK293SGnTI(-) cells, (2) lysine methylation, (3) structure-guided mutagenesis directed at better crystal packing, (4) Endo H treatment, (5) single-chain formation for stable complex, and (6) floating-drop vapor diffusion. Using our approach, the receptor-binding head domain of MV-H covalently fused with SLAM was successfully crystallized and diffraction was improved from 4.5 angstrom to a final resolution of 3.15 angstrom. These combinational methods would be useful as crystallization strategies for complexes of glycoproteins and their receptors.

Crystallization and preliminary X-ray structure analysis of human ribosomal protein L30e
Akiko Kawaguchi, Toyoyuki Ose, Min Yao, Isao Tanaka
ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, 67, 1516, 1518, WILEY-BLACKWELL, Dec. 2011, [Peer-reviewed]
English, Scientific journal, Many functions have been reported for the eukaryotic ribosomal protein L30e. L30e makes several inter-subunit and intra-subunit interactions with protein or RNA components of the 80S ribosome. Yeast L30e has been shown to bind to its own transcript to autoregulate expression at both the transcriptional and the translational levels. Furthermore, it has been reported that mammalian L30e is a component of the selenocysteine-incorporation machinery by binding to the selenocysteine-insertion sequence on mRNA. As high-resolution crystal structures of mammalian L30e are not available, the purification, crystallization and X-ray structure analysis of human L30e are presented here.

Remarkable synergistic effect between MonBI and MonBII on epoxide opening reaction in ionophore polyether monensin biosynthesis
Kyohei Sato, Atsushi Minami, Toyoyuki Ose, Hiroki Oguri, Hideaki Oikawa
TETRAHEDRON LETTERS, 52, 41, 5277, 5280, PERGAMON-ELSEVIER SCIENCE LTD, Oct. 2011, [Peer-reviewed]
English, Scientific journal, Enzymatic epoxide-opening cascade is one of the key biosynthetic processes for constructing structurally diverse polyethers. Here we report the first in vitro analysis of the cyclization catalyzed by two epoxide hydrolases, MonBI and MonBII involved in monensin biosynthesis, using simple epoxy-alcohols and the unprecedented synergistic effect on the epoxide-opening activity between these epoxide hydrolases. (C) 2011 Elsevier Ltd. All rights reserved.

Crystallographic Snapshots of Tom20-Mitochondrial Presequence Interactions with Disulfide-Stabilized Peptides
Takashi Saitoh, Mayumi Igura, Yusuke Miyazaki, Toyoyuki Ose, Nobuo Maita, Daisuke Kohda
BIOCHEMISTRY, 50, 24, 5487, 5496, AMER CHEMICAL SOC, Jun. 2011, [Peer-reviewed]
English, Scientific journal, Most mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. The Tom20 protein, residing on the mitochondrial surface, recognizes the N-terminal presequences of precursor proteins. We previously determined the crystal structures of the Tom20 presequence complex. The successful crystallization involved tethering the presequence to Tom20 through an intermolecular disulfide bond with an optimized linker. In this work, we assessed the tethering method. The intermolecular disulfide bond was cleaved in crystal with a reducing agent. The pose (i.e., conformation and position) of the presequence was identical to the previously determined pose. In another experiment, a longer linker than the optimized length was used for the tethering. The perturbation of the tether changed the pose slightly, but the interaction mode was preserved. These results argue against the forced interaction of the presequence by its covalent attachment to Tom20. Second, as an alternative method referred to as "molecular stiffening", we introduced a disulfide bond within the presequence peptide to restrict the freedom of the peptide in the unbound states. One presequence analogue exhibited over 100-fold higher affinity than its linear counterpart and generated cocrystals with Tom20. One of the two crystallographic snapshots revealed a known pose previously determined by the tethering method, and the other snapshot depicted a new pose. These results confirmed and extended the dynamic, multiple bound state model of the Tom20-presequence interactions and also demonstrated the validity of the molecular tethering and stiffening techniques in studies of transient protein-peptide interactions.

Crystallization and preliminary X-ray crystallographic study of a methyltransferase involved in 2-methylisoborneol biosynthesis in Streptomyces lasaliensis
Orapin Ariyawutthiphan, Toyoyuki Ose, Muneya Tsuda, YongGui Gao, Min Yao, Atsushi Minami, Hideaki Oikawa, Isao Tanaka
ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 67, 417, 420, WILEY-BLACKWELL, Mar. 2011, [Peer-reviewed]
English, Scientific journal, The biosynthetic pathway of the off-flavour terpenoid alcohol 2-methylisoborneol (2-MIB) requires geranyl pyrophosphate methyltransferase (GPPMT) to methylate GPP before the cyclization reaction. GPPMT is the first example of an S-adenosyl-l-methionine-dependent methyltransferase that acts on general intermediates such as geranyl pyrophosphate and farnesyl pyrophosphate in isoprenoid biosynthetic pathways. In this study, recombinant GPPMT was overproduced, purified and crystallized in the absence and presence of cofactor, cofactor analogue and substrate. Well diffracting crystals of apo GPPMT containing one molecule in the asymmetric unit were obtained and the structure of this form was solved by the molecular-replacement method. Two crystal forms of the tertiary complex with GPP and sinefungin were also obtained. Structure analysis of these crystals is currently under way in order to understand the enzyme reaction mechanism.

Structure of the measles virus hemagglutinin bound to its cellular receptor SLAM
Takao Hashiguchi, Toyoyuki Ose, Marie Kubota, Nobuo Maita, Jun Kamishikiryo, Katsumi Maenaka, Yusuke Yanagi
NATURE STRUCTURAL & MOLECULAR BIOLOGY, 18, 2, 135, U191, NATURE PUBLISHING GROUP, Feb. 2011, [Peer-reviewed]
English, Scientific journal, Measles virus, a major cause of childhood morbidity and mortality worldwide, predominantly infects immune cells using signaling lymphocyte activation molecule (SLAM) as a cellular receptor. Here we present crystal structures of measles virus hemagglutinin (MV-H), the receptor-binding glycoprotein, in complex with SLAM. The MV-H head domain binds to a beta-sheet of the membrane-distal ectodomain of SLAM using the side of its beta-propeller fold. This is distinct from attachment proteins of other paramyxoviruses that bind receptors using the top of their beta-propeller. The structure provides templates for antiviral drug design, an explanation for the effectiveness of the measles virus vaccine, and a model of the homophilic SLAM-SLAM interaction involved in immune modulations. Notably, the crystal structures obtained show two forms of the MV-H-SLAM tetrameric assembly (dimer of dimers), which may have implications for the mechanism of fusion triggering.

3D1358 The Recognition Mechanism of eIF2β for its partner proteins eIF5 and eIF2β_ε(3D Protein: Structure & Function 3,The 49th Annual Meeting of the Biophysical Society of Japan)
Gai Zuoqi, Kitagawa Yumie, Yao Min, Ose Toyoyuki, Tanaka Yoshikazu, Tanaka Isao
Seibutsu Butsuri, 51, S119, S120, The Biophysical Society of Japan General Incorporated Association, 2011
English

Importance of the Hydrogen Bonding Network Including Asp52 for Catalysis, as Revealed by Asn59 Mutant Hen Egg-white Lysozymes
Toyoyuki Ose, Kimiko Kuroki, Masaaki Matsushima, Katsumi Maenaka, Izumi Kumagai
JOURNAL OF BIOCHEMISTRY, 146, 5, 651, 657, OXFORD UNIV PRESS, Nov. 2009, [Peer-reviewed]
English, Scientific journal, In the catalysis of sugar hydrolysis by hen egg-white lysozyme, Asp52 is thought to stabilize the reaction intermediate. This residue is involved in the well-ordered hydrogen bonding network including Asn46, Asp48, Ser50 and Asn59 on the anti-parallel -sheet, designated as a platform, on which the substrate sugar sits. To reveal the role of this hydrogen bonding network in the hydrolysis, we characterized Asn59 mutants by biochemical and crystallographic studies. Surprisingly, the introduction of only a methylene group by the Asn59Gln mutation markedly reduced the bacteriolytic activity and abolished the hydrolytic activity towards the synthetic substrate, PNP-(GlcNAc)(5). A similar result was also obtained with the Asn59Asp mutant. The crystal structure of the Asn59Asp mutant in complex with the substrate analogue revealed that, as in the wild-type, the (GlcNAc)(3) was bound in the ABC subsites. The reduced activity would be caused by subtle changes in the side-chain orientations as well as the electrostatic characteristics of Asp59, resulting in the rearrangement of the hydrogen bonding network of the platform. These results suggest that the precise locations of these platform residues, maintained by the well-ordered hydrogen bonding network, are crucial for efficient hydrolysis.

The structure of alanyl-tRNA synthetase with editing domain
Masaaki Sokabe, Toyoyuki Ose, Akiyoshi Nakamura, Keita Tokunaga, Osamu Nureki, Min Yao, Isao Tanaka
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 106, 27, 11028, 11033, NATL ACAD SCIENCES, Jul. 2009, [Peer-reviewed]
English, Scientific journal, Alanyl-tRNA synthetase (AlaRS) catalyzes synthesis of Ala-tRNA(Ala) and hydrolysis of mis-acylated Ser- and Gly-tRNA(Ala) at 2 different catalytic sites. Here, we describe the monomer structures of C-terminal truncated archaeal AlaRS, with both activation and editing domains in the apo form, in complex with an Ala-AMP analog, and in a high-resolution lysine-methylated form. The structures show docking of the editing domain to the activation domain opposite from the predicted tRNA-binding surface. Thus, the editing site is positioned > 35 angstrom from the activation site, prompting us to model 2 different tRNA complexes: one binding tRNA at the activation site, and the other binding tRNA at the editing site. Interestingly, a gel-shift assay also implies the presence of 2 types of tRNA complex with different mobility. These results suggest that tRNA translocation via a canonical CCA flipping is unlikely to occur in AlaRS. The structure also demonstrated the binding of zinc in the editing site, in which the specific coordination of zinc would be facilitated by a conserved GGQ motif, implying that the editing mechanism may not be the same as in ThrRS. As Asn-194 in eubacterial AlaRS important for Ser misactivation is replaced by Thr-213 in archaeal AlaRS, a different Ser accommodation mechanism is proposed.

Tom20 recognizes mitochondrial presequences through dynamic equilibrium among multiple bound states
Takashi Saitoh, Mayumi Igura, Takayuki Obita, Toyoyuki Ose, Rieko Kojima, Katsumi Maenaka, Toshiya Endo, Daisuke Kohda
EMBO JOURNAL, 26, 22, 4777, 4787, NATURE PUBLISHING GROUP, Nov. 2007, [Peer-reviewed]
English, Scientific journal, Most mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. The N-terminal presequences of mitochondrial-precursor proteins contain a diverse consensus motif (phi chi chi phi phi, phi is hydrophobic and chi is any amino acid), which is recognized by the Tom20 protein on the mitochondrial surface. To reveal the structural basis of the broad selectivity of Tom20, the Tom20 presequence complex was crystallized. Tethering a presequence peptide to Tom20 through a disulfide bond was essential for crystallization. Unexpectedly, the two crystals with different linker designs provided unique relative orientations of the presequence with respect to Tom20, and neither configuration could fully account for the hydrophobic preference at the three hydrophobic positions of the consensus motif. We propose the existence of a dynamic equilibrium in solution among multiple states including the two bound states. In accordance, NMR N-15 relaxation analyses suggested motion on a sub-millisecond timescale at the Tom20-presequence interface. We suggest that the dynamic, multiple-mode interaction is the molecular mechanism facilitating the broadly selective specificity of the Tom20 receptor toward diverse mitochondrial presequences.

Structural basis of the 3'-end recognition of a leading strand in stalled replication forks by PriA
Kaori Sasaki, Toyoyuki Ose, Naoaki Okamoto, Katsumi Maenaka, Taku Tanaka, Hisao Masai, Mihoko Saito, Tsuyoshi Shirai, Daisuke Kohda
EMBO JOURNAL, 26, 10, 2584, 2593, NATURE PUBLISHING GROUP, May 2007, [Peer-reviewed]
English, Scientific journal, In eubacteria, PriA helicase detects the stalled DNA replication forks. This critical role of PriA is ascribed to its ability to bind to the 3' end of a nascent leading DNA strand in the stalled replication forks. The crystal structures in complexes with oligonucleotides and the combination of fluorescence correlation spectroscopy and mutagenesis reveal that the N-terminal domain of PriA possesses a binding pocket for the 3'-terminal nucleotide residue of DNA. The interaction with the deoxyribose 3'-OH is essential for the 3'-terminal recognition. In contrast, the direct interaction with 3'-end nucleobase is unexpected, considering the same affinity for oligonucleotides carrying the four bases at the 3' end. Thus, the N-terminal domain of PriA recognizes the 3'end base in a base-non-selective manner, in addition to the deoxyribose and 5'-side phosphodiester group, of the 3''-terminal nucleotide to acquire both sufficient affinity and non-selectivity to find all of the stalled replication forks generated during DNA duplication. This unique feature is prerequisite for the proper positioning of the helicase domain of PriA on the unreplicated double-stranded DNA.

Structural basis for dynamic interdomain movement and RNA recognition of the selenocysteine-specific elongation factor SelB
Toyoyuki Ose, Nicolas Soler, Linda Rasubala, Kimiko Kuroki, Daisuke Kohda, Dominique Fourmy, Satoko Yoshizawa, Katsumi Maenaka
STRUCTURE, 15, 5, 577, 586, CELL PRESS, May 2007, [Peer-reviewed]
English, Scientific journal, Selenocysteine (Sec) is the "21st" amino acid and is genetically encoded by an unusual incorporation system. The stop codon UGA becomes a Sec codon when the selenocysteine insertion sequence (SECIS) exists downstream of UGA. Sec incorporation requires a specific elongation factor, SeIB, which recognizes tRNA(Sec) via use of an EF-Tu-like domain and the SECIS mRNA hairpin via use of a C-terminal domain (SeIB-C). SeIB functions in multiple translational steps: binding to SECIS mRNA and tRNA(Sec), delivery of tRNA(Sec) onto an A site, GTP hydrolysis, and release from tRNA and mRNA. However, this dynamic mechanism remains to be revealed. Here, we report a large domain rearrangement in the structure of SeIB-C complexed with RNA. Surprisingly, the interdomain region forms new interactions with the phosphate backbone of a neighboring RNA, distinct from SECIS RNA binding. This SeIB-RNA interaction is sequence independent, possibly reflecting SeIB-tRNA/-rRNA recognitions. Based on these data, the dynamic SeIB-ribosome-mRNA-tRNA interactions will be discussed.

Crystal structure of the human monocyte-activating receptor, "Group 2" leukocyte Ig-like receptor A5 (LILRA5/LIR9/ILT11)
Mitsunori Shiroishi, Mizuho Kajikawa, Kimiko Kuroki, Toyoyuki Ose, Daisuke Kohda, Katsumi Maenaka
JOURNAL OF BIOLOGICAL CHEMISTRY, 281, 28, 19536, 19544, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Jul. 2006, [Peer-reviewed]
English, Scientific journal, Human leukocyte Ig-like receptor B1 (LILRB1) and B2 (LILRB2) belong to "Group 1" receptors and recognize a broad range of major histocompatibility complex class I molecules (MHCIs). In contrast, "Group 2" receptors show low similarity with LILRB1/B2, and their ligands remain to be identified. To date, the structural and functional characteristics of Group 2 LILRs are poorly understood. Here we report the crystal structure of the extracellular domain of LILRA5, which is an activating Group2 LILR expressed on monocytes and neutrophils. Unexpectedly, the structure showed large changes in structural conformation and charge distribution in the region corresponding to the MHCI binding site of LILRB1/B2, which are also distinct from killer cell Ig-like receptors and F alpha receptors. These changes probably confer the structural hindrance for the MHCI binding, and their key amino acid substitutions are well conserved in Group 2 LILRs. Consistently, the surface plasmon resonance and flow cytometric analyses demonstrated that LILRA5 exhibited no affinities to all tested MHCIs. These results raised the possibility that LILRA5 as well as Group 2 LILRs do not play a role in any MHCI recognition but could possibly bind to non-MHCI ligand(s) on the target cells to provide a novel immune regulation mechanism.

Efficient leukocyte Ig-like receptor signaling and crystal structure of disulfide-linked HLA-G dimer
M Shiroishi, K Kuroki, T Ose, L Rasubala, Shiratori, I, H Arase, K Tsumoto, Kumagai, I, D Kohda, K Maenaka
JOURNAL OF BIOLOGICAL CHEMISTRY, 281, 15, 10439, 10447, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Apr. 2006, [Peer-reviewed]
English, Scientific journal, HLA-G is a nonclassical major histocompatibility complex class I (MHCI) molecule, which is expressed in trophoblasts and confers immunological tolerance in the maternal-fetal interface by binding to leukocyte Ig-like receptors (LILRs, also called as LIR/ILT/CD85) and CD8. HLA-G is expressed in disulfide-linked dimer form both in solution and at the cell surface. Interestingly, MHCI dimer formations have been involved in pathogenesis and T cell activation. The structure and receptor binding characteristics of MHCI dimers have never been evaluated. Here we performed binding studies showing that the HLA-G dimer exhibited higher overall affinity to LILRB1/2 than the monomer by significant avidity effects. Furthermore, the cell reporter assay demonstrated that the dimer formation remarkably enhanced the LILRB1-mediated signaling at the cellular level. We further determined the crystal structure of the wild-type dimer of HLA-G with the intermolecular Cys(42)-Cys(42) disulfide bond. This dimer structure showed the oblique configuration to expose two LILR/CD8-binding sites upward from the membrane easily accessible for receptors, providing plausible 1: 2 (HLA-G dimer: receptors) complex models. These results indicated that the HLA-G dimer conferred increased avidity in a proper structural orientation to induce efficient LILR signaling, resulting in the dominant immunosuppressive effects. Moreover, structural and functional implications for other MHCI dimers observed in activated T cells and the pathogenic allele, HLA-B27, are discussed.

Crystallization and preliminary crystallographic analysis of the N-terminal domain of PriA from Escherichia coli.
Sasaki K, Ose T, Tanaka T, Mizukoshi T, Ishigaki T, Maenaka K, Masai H, Kohda D
Biochimica et biophysica acta, 1764, 157, 160, 1, Jan. 2006, [Peer-reviewed]

Crystallization and preliminary X-ray analysis of mitochondrial presequence receptor Tom20 in complexes with a presequence from aldehyde dehydrogenase
M Igura, T Ose, T Obita, C Sato, K Maenaka, T Endo, D Kohda
ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 61, 514, 517, BLACKWELL PUBLISHING, May 2005, [Peer-reviewed]
English, Scientific journal

Crystallization and preliminary X-ray analysis of the mRNA-binding domain of elongation factor SelB in complex with RNA
L Rasubala, D Fourmy, T Ose, D Kohda, K Maenaka, S Yoshizawa
ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 61, 296, 298, BLACKWELL PUBLISHING, Mar. 2005, [Peer-reviewed]
English, Scientific journal

Crystallization and preliminary X-ray analysis of alpha-xylosidase from Escherichia coli
M Kitamura, T Ose, M Okuyama, H Watanabe, M Yao, H Mori, A Kimura, Tanaka, I
ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 61, 178, 179, BLACKWELL PUBLISHING, Feb. 2005, [Peer-reviewed]
English, Scientific journal

Structural basis for mRNA recognition by elongation factor SelB
S Yoshizawa, L Rasubala, T Ose, D Kohda, D Fourmy, K Maenaka
NATURE STRUCTURAL & MOLECULAR BIOLOGY, 12, 2, 198, 203, NATURE PUBLISHING GROUP, Feb. 2005, [Peer-reviewed]
English, Scientific journal, In bacteria, incorporation of selenocysteine, the 21(st) amino acid, into proteins requires elongation factor SelB, which has the unusual property of binding to both transfer RNA (tRNA) and mRNA. SelB binds to an mRNA hairpin formed by the selenocysteine insertion sequence (SECIS) with extremely high specificity, the molecular basis of which has been unknown. We have determined the crystal structure of the mRNA-binding domain of SelB in complex with SECIS RNA at a resolution of 2.3 Angstrom. This is the first example of a complex between an RNA and a winged-helix (WH) domain, a motif found in many DNA-binding proteins and recently discovered in RNA-binding proteins. Notably, RNA binding does not induce a major conformational change in the WH motif. The structure reveals a new mode of RNA recognition with a geometry that allows the complex to wrap around the small ribosomal subunit.

Crystal structure of a biologically functional form of PriB from Escherichia coli reveals a potential single-stranded DNA-binding site
S Shioi, T Ose, K Maenaka, M Shiroishi, Y Abe, D Kohda, T Katayama, T Ueda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 326, 4, 766, 776, ACADEMIC PRESS INC ELSEVIER SCIENCE, Jan. 2005, [Peer-reviewed]
English, Scientific journal, PriB is not only an essential protein necessary for the replication restart on the collapsed and disintegrated replication fork, but also an important protein for assembling of primosome onto PhiX174 genomic DNA during replication initiation. Here we report a 2.0-Angstrom-resolution X-ray structure of a biologically functional form of PriB from Escherichia coli. The crystal structure revealed that despite a low level of primary sequence identity, the PriB monomer, as well as the dimeric form, are structurally identical to the N-terminal DNA-binding domain of the single-stranded DNA-binding protein (SSB) from Escherichia coli, which possesses an oligonucleotides-binding-fold. The oligonucleotide-PriB complex model based on the oligonucleotides-SSB complex structure suggested that PriB had a DNA-binding pocket conserved in SSB from Escherichia coli and might bind to single-stranded DNA in the manner of SSB. Furthermore, surface plasmon resonance analysis and fluorescence measurements demonstrated that PriB binds single-stranded DNA with high affinity, by involving tryptophan residue. The significance of these results with respect to the functional role of PriB in the assembly of primosome is discussed. (C) 2004 Published by Elsevier Inc.

Crystal structure of the ATP-binding cassette of multisugar transporter from Pyrococcus horikoshii OT3
T Ose, T Fujie, M Yao, N Watanabe, Tanaka, I
PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS, 57, 3, 635, 638, WILEY-LISS, Nov. 2004, [Peer-reviewed]
English, Scientific journal

Structural and enzymatic properties of 1-aminocyclopropane-1-carboxylate deaminase homologue from Pyrococcus horikoshii
A Fujino, T Ose, M Yao, T Tokiwano, M Honma, N Watanabe, Tanaka, I
JOURNAL OF MOLECULAR BIOLOGY, 341, 4, 999, 1013, ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD, Aug. 2004, [Peer-reviewed]
English, Scientific journal, 1-Aminocyclopropane-1-carboxylate deaminase (ACCD) is a pyridoxal 5'-phosphate dependent enzyme that shows deaminase activity toward ACC, a precursor of plant hormone ethylene. ACCD from some soil bacteria has been reported to be able to break the cyclopropane ring of ACC to yield alpha-ketobutyrate and ammonia. We reported the crystal structure of ACCD from the yeast Hansenula saturnus in the absence/presence of substrate ACC, and proposed its ingenious reaction mechanisms. In order to study the enzyme further, we overexpressed the ACCD homologue protein (phAHP) from the fully decoded hyperthermophilic archearon, Pyrococcus horikoshii OT3. However, phAHP does not show ACCD activity at high temperature as well as at room temperature, though it has significant sequence similarity. Instead of ACCD activity the GC-MS analysis and enzymatic method show that phAHP has deaminase activity toward L and D-serine. Here, we present the crystal structures of the native and ACC-complexed phAHP. The overall topology of the phAHP structure is very similar to that of ACCD; however, critical differences were observed around the active site. Here, the differences of enzymatic activity between phAHP and ACCD are discussed based on the structural differences of these two proteins. We suggest that the catalytic disagreement between these two enzymes comes from the difference of the residues near the pyridine ring of pyridoxal 5'-phosphate (PLP), not the difference of the catalytic residues themselves. We also propose a condition necessary in the primary sequence to have ACCD activity. (C) 2004 Elsevier Ltd. All rights reserved.

Structure of macrophomate synthase
T Ose, K Watanabe, M Yao, M Honma, H Oikawa, Tanaka, I
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 60, 1187, 1197, BLACKWELL MUNKSGAARD, Jul. 2004, [Peer-reviewed]
English, Scientific journal, Macrophomate synthase (MPS) is an enzyme that catalyzes an extraordinarily complex conversion reaction, including two decarboxylations, two carbon - carbon bond formations and a dehydration, to form the benzoate analogue macrophomate from a 2-pyrone derivative and oxalacetate. Of these reactions, the two carbon - carbon bond formations are especially noteworthy because previous experiments have indicated that they proceed via a Diels - Alder reaction, one of the most widely used reactions in organic synthesis. The structural evidence that MPS catalyzes an intermolecular Diels - Alder reaction has been reported recently [Ose et al. ( 2003), Nature ( London), 422, 185 - 189]. Interestingly, the tertiary structure as well as the quaternary structure of MPS are similar to those of 2-dehydro-3-deoxygalactarate (DDG) aldolase, a carbon - carbon bond-forming enzyme that catalyzes the reversible reaction of aldol condensation/cleavage. Here, the structure of MPS is described in detail and compared with that of DDG aldolase. Both enzymes have a (beta/alpha)(8)-barrel fold and are classified as belonging to the enolase superfamily based on their reaction strategy. The basic principles for carbon - carbon bond formation used by both MPS and DDG aldolase are the same with regard to trapping the enolate substrate and inducing subsequent reaction. The major differences in the active sites between these two enzymes are the recognition mechanisms of the second substrates, 2-pyrone and DDG, respectively.

3SB03 Diels-Alder Reaction in Proteins
Ose T.
Seibutsu Butsuri, 44, S20, The Biophysical Society of Japan General Incorporated Association, 2004
Japanese

Reaction intermediate structures of 1-aminocyclopropane-1-carboxylate deaminase - Insight into PLP-dependent cyclopropane ring-opening reaction
T Ose, A Fujino, M Yao, N Watanabe, M Honma, Tanaka, I
JOURNAL OF BIOLOGICAL CHEMISTRY, 278, 42, 41069, 41076, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Oct. 2003, [Peer-reviewed]
English, Scientific journal, The pyridoxal 5'-phosphate-dependent enzymes have been evolved to catalyze diverse substrates and to cause the reaction to vary. 1-Aminocyclopropane-1-carboxylate deaminase catalyzes the cyclopropane ring-opening reaction followed by deamination specifically. Since it was discovered in 1978, the enzyme has been widely investigated from the mechanistic and physiological viewpoints because the substrate is a precursor of the plant hormone ethylene and the enzymatic reaction includes a cyclopropane ring-opening. We have previously reported the crystal structure of the native enzyme. Here we report the crystal structures of the two reaction intermediates created by the mutagenesis complexed with the substrate. The substrate was validated in the active site of two forms: 1) covalent-bonded external aldimine with the coenzyme in the K51T form and 2) the non-covalent interaction around the coenzyme in the Y295F form. The orientations of the substrate in both structures were quite different form each other. In concert with other site-specific mutation experiments, this experiment revealed the ingenious and unique strategies that are used to achieve the specific activity. The substrate incorporated into the active site is reactivated by a two-phenol charge relay system to lead to the formation of a Schiff base with the coenzyme. The catalytic Lys(51) residue may play a novel role to abstract the methylene proton from the substrate in cooperation with other factors, the carboxylate group of the substrate and the electron-adjusting apparatuses of the coenzyme.

Insight into a natural Diels-Alder reaction from the structure of macrophomate synthase
T Ose, K Watanabe, T Mie, M Honma, H Watanabe, M Yao, H Oikawa, Tanaka, I
NATURE, 422, 6928, 185, 189, NATURE PUBLISHING GROUP, Mar. 2003, [Peer-reviewed]
English, Scientific journal, The Diels-Alder reaction, which forms a six-membered ring from an alkene (dienophile) and a 1,3-diene, is synthetically very useful for construction of cyclic products with high regio- and stereoselectivity under mild conditions(1). It has been applied to the synthesis of complex pharmaceutical and biologically active compounds(2). Although evidence(3-7) on natural Diels-Alderases has been accumulated in the biosynthesis of secondary metabolites(8), there has been no report on the structural details of the natural Diels-Alderases. The function and catalytic mechanism of the natural Diels-Alderase are of great interest owing to the diversity of molecular skeletons in natural Diels-Alder adducts(8). Here we present the 1.70 Angstrom resolution crystal structure of the natural Diels-Alderase, fungal macrophomate synthase (MPS)(3), in complex with pyruvate. The active site of the enzyme is large and hydrophobic, contributing amino acid residues that can hydrogen-bond to the substrate 2-pyrone. These data provide information on the catalytic mechanism of MPS, and suggest that the reaction proceeds via a large-scale structural reorganization of the product.

Catalytic activity analysis of PLP dependent enzyme PH0054 from hyperthermophilic archaebacteria P.horikoshii OT3 by X-ray crystallography
Fujino A., Ose T., Yao M., Honma M., Tanaka I.
Seibutsu Butsuri, 41, S100, The Biophysical Society of Japan General Incorporated Association, 2001
Japanese

Crystal structure of 1-aminocyclopropane-1-carboxylate deaminase from Hartsenula saturnus
M Yao, T Ose, H Sugimoto, A Horiuchi, A Nakagawa, S Wakatsuki, D Yokoi, T Murakami, M Honma, Tanaka, I
JOURNAL OF BIOLOGICAL CHEMISTRY, 275, 44, 34557, 34565, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Nov. 2000, [Peer-reviewed]
English, Scientific journal, 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 PUP-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 Angstrom 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.

Reaction and structure of 1-aminocyclopropane-1-carboxylate deaminase
M Honma, T Murakami, T Ose, H Matsui, Tanaka, I
BIOCHEMISTRY AND MOLECULAR BIOLOGY OF VITAMIN B6 AND PQQ-DEPENDENT PROTEINS, 171, 174, BIRKHAUSER VERLAG AG, 2000, [Peer-reviewed]
English, International conference proceedings, 1-Aminocyclopropane-1-carboxylate (ACC) deaminase was isolated from fungi Hansenula saturnus and Penicillium citricum, and their cDNA were analyzed. Amino acid sequences of the enzymes from H. saturnus and P. citrinum showed 61% and 52% of similarity respectively to that of a bacterial enzyme. Furthermore, the alignment of these sequences indicates their similarity to the tryptophan synthase beta -subunit. This became clearly evident when crystallographical analysis of ACC deaminase from H. saturnus was conducted. From the three-dimensional figure, three residues found near the PLP were chosen for preparation of the mutant enzymes.

植物ホルモンの生合成を制御する酵素ACCDの構造解析
姚 閔, 杉本 宏, 尾瀬 農之, 堀内 篤, 中川 敦史, 田中 勲, 若槻 荘市, 横井 大輔, 邑上 農隆, 本間 守
日本結晶学会誌, 40, 48, 48, The Crystallographic Society of Japan, 1998
Japanese

X線小角散乱(SEC-SAXS)による,Breast tumor kinaseの活性化機構の解明
宇賀神魁, 武井梓穂, 松田正, 尾瀬農之, 日本蛋白質科学会年会プログラム・要旨集, 23rd (CD-ROM), 2023

アダプター蛋白質介在に伴う,乳がん特異的キナーゼの活性亢進評価
武井梓穂, 宇賀神魁, 中迫純希, 松尾友樹, 神田諒, 前仲勝実, 松田正, 姚閔, 尾瀬農之, 日本蛋白質科学会年会プログラム・要旨集, 23rd (CD-ROM), 2023

新規酵素ガラクトース2-エピメラーゼの諸性質ならびにX線結晶構造の解析
内山昌典, 佐分利亘, 武井梓穂, 尾瀬農之, 森春英, 日本農芸化学会北海道支部学術講演会講演要旨集(Web), 2023, 2023

狂犬病ウイルスPタンパク質によるSTAT阻害機構の解明
南未来, 杉山葵, 武川祐一郎, 姚閔, 姚閔, 廣瀬未果, 杉田征彦, 尾瀬農之, 尾瀬農之, 日本蛋白質科学会年会プログラム・要旨集, 23rd (CD-ROM), 2023

世界初IDP酵素の検証
大和田ゆうき, 澤田光平, 荒井彩花, 田所高志, 南篤志, 前仲勝実, 久米田博之, 姚閔, 及川英秋, 尾瀬農之, 尾瀬農之, 日本結晶学会年会講演要旨集, 2022, 2022

Structure determination of protein-tRNA complex using AlphaFold2 predicted model
碓井拓哉, 大江花, 中村彰良, 尾瀬農之, 尾瀬農之, YAO Min, YAO Min, Photon Factory News, 40, 3, 2022

狂犬病ウイルスP蛋白質による宿主の免疫経路阻害機構の解明
南未来, 杉山葵, 野間井智, JIANG Xinxin, 前仲勝実, YAO Min, 尾瀬農之, 日本蛋白質科学会年会プログラム・要旨集, 21st, 2021

病原性理解のためのLyssavirus P蛋白質C末端ドメイン構造解析
杉山葵, 野間井智, JIANG Xinxin, 南未来, 前仲勝実, 伊藤直人, GOOLEY Paul R., MOSELEY Gregory W., YAO Min, 尾瀬農之, 尾瀬農之, 日本蛋白質科学会年会プログラム・要旨集, 21st, 2021

麻疹ウイルスVタンパク質によるType I IFN経路阻害機構の解明
木本円花, 永野悠馬, 杉山葵, 姚閔, 田所高志, 前仲勝実, 尾瀬農之, 日本結晶学会年会講演要旨集, 2021, 2021

毒ヘビ由来Ca²⁺チャネル阻害型神経毒と毒ヘビ血清蛋白質の複合体構造
田所高志, 岡部由紀, 松原永季, 喜多俊介, 尾瀬農之, 黒木喜美子, 前仲勝実, 寺田成之, 塩井(青木)成留実, 日本結晶学会年会講演要旨集, 2021, 2021

A straightforward approach to antibodies recognising cancer specific glycopeptidic neoepitopes (vol 11, pg 4999, 2020)
Hajime Wakui, Yoshikazu Tanaka, Toyoyuki Ose, Isamu Matsumoto, Koji Kato, Yao Min, Taro Tachibana, Masaharu Sato, Kentaro Naruchi, Fayna Garcia Martin, Hiroshi Hinou, Shin-Ichiro Nishimura, CHEMICAL SCIENCE, 11, 46, 12588, 12589, Dec. 2020
Correction for 'A straightforward approach to antibodies recognising cancer specific glycopeptidic neoepitopes' by Hajime Wakui et al., Chem. Sci., 2020, 11, 4999-5006, DOI: ; 10.1039/D0SC00317D., ROYAL SOC CHEMISTRY, English, Others

Elucidation of PLP binding and substrate recognition mechanism of plant tyrosine decarboxylase
WANG Hang, NAKAGAWA Yusuke, SATOH Yasuharu, YU Jian, OSE Toyoyuki, YAO Min, 量子ビームサイエンスフェスタ(Web), 2019, 2020

リッサウイルスP蛋白質C末端ドメインの構造比較
杉山葵, 野間井智, 蒋欣欣, 南未来, 前仲勝実, 伊藤直人, GOOLEY Paul R., MOSELEY Gregory W., 姚閔, 尾瀬農之, 尾瀬農之, 日本結晶学会年会講演要旨集, 2020 (CD-ROM), 2020

Crystal structure analysis of the C-terminal domain of Duvenhage virus P protein
杉山葵, 蒋欣欣, 前仲勝実, 姚閔, 尾瀬農之, 量子ビームサイエンスフェスタ(Web), 2019, 2020

バクテリアセルロース合成酵素複合体構成成分BosCの構造解析の試み
佐藤亨, 大内香予子, 今井友也, 田島健次, 尾瀬農之, 姚閔, 日本結晶学会年会講演要旨集, 2020 (CD-ROM), 2020

CD1dとアミド基導入抗原複合体のX線結晶構造解析　　　　　　　　　　　　　　　
喜多 俊介, 日下 裕規, 井貫 晋輔, Md. Imran Hossain, 花島 慎弥, 田所 高志, 新山 真由美, 杉山 成, 相羽 俊彦, 尾瀬 農之, 黒木 喜美子, 深瀬 浩一, 村田 道雄, 藤本 ゆかり, 前仲 勝実, 日本結晶学会 講演要旨集, Nov. 2019

Crystal structure analysis of lipid presenting molecule CD1d
喜多俊介, 日下裕規, HOSSAIN Md. Imran, 花島慎弥, 井貫晋輔, 田所高志, 新山真由美, 杉山成, 相羽俊彦, 相羽俊彦, 尾瀬農之, 黒木喜美子, 深瀬浩一, 藤本ゆかり, 村田道雄, 前仲勝実, 量子ビームサイエンスフェスタ(Web), 2018, 2019

イヌジステンパーウイルス受容体結合タンパク質の構造と膜融合の活性化機構
福原秀雄, 酒匂幸, 河村美尋, 梶川瑞穂, 橋口隆生, 竹田誠, PHILIPPE Plattet, 尾瀬農之, 前仲勝実, 日本ウイルス学会学術集会プログラム・予稿集(Web), 67th, 2019

狂犬病ウイルスP蛋白質によるJAK‐STATシグナル阻害機構の解明
杉山葵, 蒋欣欣, 永野悠馬, 野間井智, 若原拓也, 前仲勝実, 姚閔, MOSLEY Gregory, 尾瀬農之, 日本細胞生物学会大会(Web), 71st, ROMBUNNO.2P‐005 (WEB ONLY), 2019
Japanese

IDPを使用した新規ペア型酵素の戦略
澤田光平, 田所高志, 大和田ゆうき, 南篤志, 久米田博之, 斎尾智英, 姚閔, 及川英秋, 前仲勝実, 尾瀬農之, 尾瀬農之, 日本結晶学会年会講演要旨集, 2018, 32, 01 Nov. 2018
Japanese

エポキシド加水分解機能を持つタンパク質の分子進化的考察　　　　　　　　　　　　　　　
飯淵 友直, 尾瀬 農之, 前仲 勝実, 及川 英秋, 園山 正史, 向井 有理, 日本生化学会大会プログラム・講演要旨集, 91回, [2P, 110], Sep. 2018
(公社)日本生化学会, Japanese

脂質抗原提示分子CD1dによる抗原認識機構の解析　　　　　　　　　　　　　　　
日下 裕規, 喜多 俊介, 大野 祐介, 木原 章雄, 尾瀬 農之, 黒木 喜美子, 前仲 勝実, 日本生化学会大会プログラム・講演要旨集, 91回, [3P, 090], Sep. 2018
(公社)日本生化学会, Japanese

エポキシド加水分解機能を持つタンパク質の分子進化的考察　　　　　　　　　　　　　　　
飯淵 友直, 尾瀬 農之, 前仲 勝実, 及川 英秋, 園山 正史, 向井 有理, 日本生化学会大会プログラム・講演要旨集, 91回, [2P, 110], Sep. 2018
(公社)日本生化学会, Japanese

脂質抗原提示分子CD1dによる抗原認識機構の解析　　　　　　　　　　　　　　　
日下 裕規, 喜多 俊介, 大野 祐介, 木原 章雄, 尾瀬 農之, 黒木 喜美子, 前仲 勝実, 日本生化学会大会プログラム・講演要旨集, 91回, [3P, 090], Sep. 2018
(公社)日本生化学会, Japanese

脂質抗原提示分子CD1dによる抗原認識における種差
日下裕規, 喜多俊介, HOSSAIN Imran, 花島慎弥, 井貫晋輔, 新山真由美, 杉山成, 相羽俊彦, 尾瀬農之, 黒木喜美子, 深瀬浩一, 藤本ゆかり, 村田道雄, 前仲勝実, 日本蛋白質科学会年会プログラム・要旨集, 18th, 84, 23 May 2018
Japanese

毒ヘビ由来Ca2+チャネル阻害型神経毒と毒ヘビ血清蛋白質の複合体構造
田所高志, 塩井(青木, 成留実, 岡部由紀, 松原永季, 喜多俊介, 尾瀬農之, 黒木喜美子, 前仲勝実, 寺田成之, 日本蛋白質科学会年会プログラム・要旨集, 18th, 90, 23 May 2018
Japanese

ヒトの自然免疫系を阻害する麻疹ウイルスV蛋白質の機能解析
永野悠馬, 若原拓也, 秦玉瑩, 柳雄介, 前仲勝実, 尾瀬農之, 尾瀬農之, 日本蛋白質科学会年会プログラム・要旨集, 18th, 64, 23 May 2018
Japanese

狂犬病ウイルスP蛋白質によるJAK‐STATシグナル阻害機構の解明
杉山葵, JIANG Xinxin, 永野悠馬, 野間井智, 若原拓也, 前仲勝実, YAO Min, MOSLEY Gregory, 尾瀬農之, 日本蛋白質科学会年会プログラム・要旨集, 18th, 48, 23 May 2018
Japanese

PILRαによる糖ペプチド認識機構の解明　　　　　　　　　　　　　　　
古川 敦, 柿田 浩輔, 荒瀬 尚, 穴田 仁洋, 尾瀬 農之, 橋本 俊一, 前仲 勝実, 日本薬学会年会要旨集, 138年会, 2, 261, 261, Mar. 2018
(公社)日本薬学会, Japanese

PILRαによる糖ペプチド認識機構の解明
古川敦, 柿田浩輔, 荒瀬尚, 穴田仁洋, 尾瀬農之, 橋本俊一, 前仲勝実, 日本薬学会年会要旨集(CD-ROM), 138th, 2, ROMBUNNO.27PA‐am327, 261, Mar. 2018
(公社)日本薬学会, Japanese

Crystal structure and functional analyses of Rabies virus P protein mutants
野間井智, 蒋欣欣, 永野悠馬, 杉山葵, 姚閔, GOOLEY Paul, MOSELEY Gregory, 前仲勝実, 尾瀬農之, 尾瀬農之, 量子ビームサイエンスフェスタ(Web), 2017, 2018

ヒト免疫受容体NKRP1AによるリガンドLLT1の分子認識機構
田所高志, 喜多俊介, 松原永季, 笠井宣征, 玉置貴晴, 岡部由紀, 日下裕規, 石山夢美, 福原秀雄, 上敷領淳, 尾瀬農之, 黒木喜美子, 前仲勝実, 日本生化学会大会(Web), 2017年度, [2P, 0185], Dec. 2017
生命科学系学会合同年次大会運営事務局, Japanese

HIV-2 Nefタンパク質のX線結晶構造　　　　　　　　　　　　　　　
平尾 憲吾, 黒木 喜美子, Andrews Sophie, 尾瀬 農之, Rowland-Jones Sarah, 前仲 勝実, 生命科学系学会合同年次大会, 2017年度, [2P, 0091], Dec. 2017
生命科学系学会合同年次大会運営事務局, English

単純ヘルペスウイルス由来糖蛋白質がヒト受容体を認識するメカニズム
尾瀬農之, 石塚幹広, 古川敦, 黒木喜美子, 前仲勝実, 日本結晶学会年会講演要旨集, 2017, 47, 23 Nov. 2017
Japanese

狂犬病ウイルスの細胞吸着に関与する宿主因子の解析
佐々木道仁, アニンディタ パウリナ, 伊藤直人, 杉山誠, 福原秀雄, 尾瀬農之, 尾瀬農之, 前仲勝実, 澤洋文, 澤洋文, 日本獣医学会学術集会講演要旨集, 160th, 400, 400, 30 Aug. 2017
(公社)日本獣医学会, Japanese

狂犬病ウイルスの細胞吸着に関与する宿主因子の解析　　　　　　　　　　　　　　　
佐々木 道仁, アニンディ・タパウリナ, 伊藤 直人, 杉山 誠, 福原 秀雄, 尾瀬 農之, 前仲 勝実, 澤 洋文, 日本獣医学会学術集会講演要旨集, 160回, 400, 400, Aug. 2017
(公社)日本獣医学会, Japanese

麻疹ウイルスV蛋白質C末端領域の構造的特徴
永野悠馬, 若原拓也, 蒋欣欣, 柳雄介, 前仲勝実, 尾瀬農之, 日本蛋白質科学会年会プログラム・要旨集, 17th, 48, 22 May 2017
Japanese

分泌タンパク質ANGPTL2を介したシグナル伝達を阻害可能な低分子化合物探索
荒牧峻彦, 黒木喜美子, 門松毅, 尾池雄一, 尾瀬農之, 前仲勝実, 日本蛋白質科学会年会プログラム・要旨集, 17th, 147, 22 May 2017
Japanese

トラスツズマブ一本鎖Fv抗体断片の構築および高機能化
田所高志, 中村光太, 坪井晴美, 前田龍, 尾瀬農之, 松田彰, 前仲勝実, 日本蛋白質科学会年会プログラム・要旨集, 17th, 148, 22 May 2017
Japanese

CD1dの一本鎖化による安定化と結晶構造解析
日下裕規, 喜多俊介, HOSSAIN Imuran Md, 花島慎弥, 井貫晋輔, 新山真由美, 杉山成, 尾瀬農之, 黒木喜美子, 藤本ゆかり, 村田道雄, 前仲勝実, 日本蛋白質科学会年会プログラム・要旨集, 17th, 120, 22 May 2017
Japanese

インフルエンザウイルス細胞侵入において鍵となる宿主タンパク質の同定　　　　　　　　　　　　　　　
藤岡 容一朗, 西出 真也, 尾瀬 農之, 加藤 いづみ, 福原 秀雄, 藤岡 真理, 堀内 浩水, 佐藤 絢, Nepal Prabha, 柏木 彩花, Wang Jing, 堀口 美香, Paudel Sarad, 南保 明日香, 宮崎 忠昭, 前仲 勝実, 大場 雄介, 日本細胞生物学会大会講演要旨集, 69回, 61, 61, May 2017
(一社)日本細胞生物学会, Japanese

インフルエンザウイルス細胞侵入において鍵となる宿主タンパク質の同定
藤岡容一朗, 西出真也, 尾瀬農之, 加藤いづみ, 福原秀雄, 藤岡真理, 堀内浩水, 佐藤絢, NEPAL Prabha, 柏木彩花, WANG Jing, 堀口美香, PAUDEL Sarad, 南保明日香, 宮崎忠昭, 前仲勝実, 前仲勝実, 大場雄介, 日本細胞生物学会大会(Web), 69th, ROMBUNNO.T8‐04(P2‐028) (WEB ONLY), 61, May 2017
(一社)日本細胞生物学会, Japanese

Insight into the activation mechanism of Brk
松尾友樹, 神田諒, 西條慎也, 清水伸隆, 前仲勝実, 尾瀬農之, 量子ビームサイエンスフェスタ(Web), 2016, 2017

自然免疫受容体Mincleの糖脂質認識機構
古川敦, 須知佑介, 池野里紗, 松丸尊紀, 上敷領淳, 齊藤貴士, 尾瀬農之, 山崎晶, 前仲勝実, 日本薬学会年会要旨集(CD-ROM), 137th, ROMBUNNO.S12‐1, 2017
Japanese

アンジオポエチン様因子Angptl2の結晶構造解析および免疫受容体LILRB2との相互作用解析
荒牧峻彦, 黒木喜美子, 門松毅, 寺田和豊, 尾池雄一, 尾瀬農之, 前仲勝実, 日本蛋白質科学会年会プログラム・要旨集, 16th, 104, 19 May 2016
Japanese

I型IFNシグナルを回避する麻疹ウイルスVタンパク質の分子基盤解析
永野悠馬, 若原拓也, 野間井智, 市川聡, 柳雄介, 前仲勝実, 尾瀬農之, 日本蛋白質科学会年会プログラム・要旨集, 16th, 85, 19 May 2016
Japanese

ヘパラン硫酸を介した狂犬病ウイルスの細胞吸着機構の解析
佐々木道仁, アニンディタ パウリナ, 伊藤直人, 杉山誠, 福原秀雄, 尾瀬農之, 前仲勝実, 大場靖子, 澤洋文, 澤洋文, 日本分子生物学会年会プログラム・要旨集(Web), 39th, ROMBUNNO.2P‐0314 (WEB ONLY), 2016
Japanese

狂犬病ウイルスGタンパク質特異的抗体の中和機構の解明
有坂知朗, 荒牧峻彦, 青木亨丞, 伊藤直人, 杉山誠, 尾瀬農之, 福原秀雄, 前仲勝実, 日本分子生物学会年会プログラム・要旨集(Web), 39th, ROMBUNNO.1P‐0598 (WEB ONLY), 2016
Japanese

構造分子生物学・生化学の進展 糖脂質認識C型レクチン受容体Mincleの構造解析と新規アジュバント探索　　　　　　　　　　　　　　　
古川 敦, 上敷領 淳, 須知 裕介, 池野 里紗, 松丸 尊紀, 児玉 耕太, 尾瀬 農之, 山崎 晶, 前仲 勝実, 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 88回・38回, [3W7, 6], Dec. 2015
(公社)日本生化学会, English

Lectin‐like transcript1(LLT1)C型レクチン様ドメインの結晶構造解析
喜多俊介, 田所高志, 松原永季, 笠井宣征, 玉置貴晴, 岡部由紀, 日下裕規, 石山夢美, 福原秀雄, 上敷領淳, KRAYUKHINA Elena, KRAYUKHINA Elena, 内山進, 尾瀬農之, 黒木喜美子, 前仲勝実, 日本生化学会大会(Web), 88回・38回, [3T17p, 02(3P0328)], Dec. 2015
(公社)日本生化学会, Japanese

構造生物学が推進する医薬基盤とその応用 抑制型免疫受容体PILRαによるヘルペスウイルス1型由来O型糖鎖およびペプチド配列の同時認識と創薬
尾瀬農之, 古川敦, 黒木喜美子, 前仲勝実, 医学のあゆみ, 254, 8, 559, 565, Aug. 2015
ウイルスは、体内・細胞侵入から放出に至るまでその生活環のすべてにおいて宿主の生命活動を巧妙に利用している。宿主因子とウイルス分子の相互作用を原子レベルで解明できれば、宿主とウイルスが攻防を繰り広げている進化の過程を目の当たりにし、感染防御に欠かせない宿主の生命現象を深く理解することができる。本研究では、単純ヘルペスウイルスI型(HSV-I)がヒト細胞に侵入する際に免疫系受容体paired Ig-like type 2レセプターα(PILRα)を利用する仕掛けを、構造生物学的手法により明らかにした。PILRαはHSV-1の細胞融合タンパク質gBの特定領域を認識するが、この認識は、修飾されたO型糖鎖と糖鎖が結合しているポリペプチド鎖を同時に認識する新奇なものであった。O型糖鎖とペプチドの両者がgBとPILRαの結合に必要なことを、表面プラズモン法により確認し、実際のウイルス感染も阻害されることが明らかとなった。この発見を利用し、HSV-I感染予防などの創薬に向けてスクリーニングや化合物の設計・合成を行っている。(著者抄録), 医歯薬出版(株), Japanese

ポリエーテル化合物生合成酵素の結晶構造解析
尾瀬農之, 笈川あずさ, 野間井智, 澤田光平, 道仙卓也, 南篤志, 及川英秋, 前仲勝実, 日本蛋白質科学会年会プログラム・要旨集, 15th, 67, 26 May 2015
Japanese

単純ヘルペスウイルス侵入阻害糖ペプチドの最適化を目指した熱力学的相互作用解析およびX線結晶構造解析
山田友樹, 古川敦, 柿田浩輔, 坂本二郎, 前田直良, 逢坂文那, 斉藤貴士, 黒木喜美子, 荒瀬尚, 穴田仁洋, 尾瀬農之, 橋本俊一, 前仲勝実, 日本蛋白質科学会年会プログラム・要旨集, 15th, 90, 26 May 2015
Japanese

JAK‐STAT経路を阻害するウイルスタンパク質の機能・構造解析
永野悠馬, 野間井智, 前仲勝実, 柳雄介, 尾瀬農之, 日本蛋白質科学会年会プログラム・要旨集, 15th, 88, 26 May 2015
Japanese

アンジオポエチン様因子Angptl2の発現系構築と免疫受容体LILRB2との相互作用解析
荒牧峻彦, 黒木喜美子, 門松毅, 寺田和豊, 尾池雄一, 尾瀬農之, 前仲勝実, 日本蛋白質科学会年会プログラム・要旨集, 15th, 138, 26 May 2015
Japanese

単純ヘルペスウイルスがペア型レセプターPILRαを利用するメカニズム
尾瀬農之, 黒木喜美子, 山口宗親, 田畑栄一, 真板宣夫, 梶川瑞穂, 中村聖子, WANG Jing, 佐藤毅史, 荒瀬尚, 前仲勝実, 物構研サイエンスフェスタ要旨集, 3rd, 79, 2015
Japanese

Mechanisms of lyssavirus evasion of innate immunity and roles in lethal rabies disease
Linda Wiltzer, Aaron Brice, Kim Lieu, Angela Harrison, Sibil Oksayan, Toby Bell, Donna Whelan, Alamgir Hossain, Patrick Shillling, Kazuma Okada, Satoko Yamaoka, Florence Larrous, Herve Bourhy, Toyoyuki Ose, Paul Gooley, David A. Jans, Danielle Blondel, Naoto Ito, Gregory Moseley, CYTOKINE, 70, 1, 60, 60, Nov. 2014
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, English, Summary international conference

単純ヘルペスウイルス1型感染阻害物質の探索
前田直良, 古川敦, 坂本二郎, 黒木喜美子, 尾瀬農之, 柿田浩輔, 穴田仁洋, 橋本俊一, 有井潤, 加藤哲久, 川口寧, 荒瀬尚, 前仲勝実, 日本ウイルス学会学術集会プログラム・抄録集, 62nd, 273, 31 Oct. 2014
Japanese

Structural Analysis for the Interaction of sialic T antigen glycopeptide of HSV-1 with Entry Receptor PILR alpha
Takao Nomura, Jiro Sakamoto, Fumina Oosaka, Kosuke Kakita, Atsushi Furukawa, Masahiro Anada, Shunichi Hashimoto, Kimiko Kuroki, Toyoyuki Ose, Hisashi Arase, Takashi Saitoh, Katsumi Maenaka, PROTEIN SCIENCE, 23, 230, 230, Jul. 2014
WILEY-BLACKWELL, English, Summary international conference

C型レクチン様受容体MincleとMCLの糖脂質認識機構
古川敦, 上敷領淳, 森大輝, 豊永憲司, 岡部由紀, 藤司亜也, 神田諒, 三宅靖延, 尾瀬農之, 山崎晶, 前仲勝実, 日本蛋白質科学会年会プログラム・要旨集, 14th, 121, 26 May 2014
Japanese

2SBP-02 Structural basis for glycolipid recognition mechanism by C-type lectin like receptor, Mincle(New Development of Structural Cell Biology in Signal Transduction,Symposium,The 52th Annual Meeting of the Biophysical Society of Japan(BSJ2014))
Furukawa Atsushi, Kamishikiryo Jun, Ose Toyoyuki, Yamasaki Sho, Maenaka Katsumi, Seibutsu Butsuri, 54, 1, S131, 2014
一般社団法人 日本生物物理学会, English

単純ヘルペスウイルスがペア型レセプターPILRαを利用する分子基盤
尾瀬農之, 黒木喜美子, 山口宗親, 田畑栄一, 真板宣夫, 梶川瑞穂, 中村聖子, WANG Jing, 佐藤毅史, 荒瀬尚, 前仲勝実, 日本結晶学会年会講演要旨集, 2014, 110, 2014
Japanese

Structure analysis of geranyl pyrophosphate methyltransferase and the proposed reaction mechanism of SAM-dependent C-methylation (vol D68, pg 1558, 2012)
Orapin Ariyawutthiphan, Toyoyuki Ose, Atsushi Minami, Sandip Shinde, Muneya Tsuda, Yong-Gui Gao, Min Yao, Hideaki Oikawa, Isao Tanaka, ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 69, 2584, 2584, Dec. 2013
WILEY-BLACKWELL, English, Others

monensin生合成酵素の結晶構造解析
道仙卓也, 南篤志, 笈川あずさ, 佐藤恭平, 前仲勝実, 及川英秋, 尾瀬農之, 日本結晶学会年会講演要旨集, 2013, 75, 12 Oct. 2013
Japanese

MurDの大量発現系の構築と結晶化
笈川あずさ, 北辻千展, 尾瀬農之, 前仲勝実, 日本蛋白質科学会年会プログラム・要旨集, 13th, 123, 31 May 2013
Japanese

C型レクチン様受容体Mindeの糖脂質認識機構
古川敦, 上敷領淳, 岡部由紀, 神田涼, 豊永憲司, 森大輝, 三宅靖延, 山崎晶, 尾瀬農之, 前仲勝実, 日本蛋白質科学会年会プログラム・要旨集, 13th, 115, 31 May 2013
Japanese

モネンシン生合成酵素の結晶構造解析
尾瀬農之, 笈川あずさ, 南篤志, 佐藤恭平, 及川英秋, 前仲勝実, 日本蛋白質科学会年会プログラム・要旨集, 13th, 122, 31 May 2013
Japanese

樹状細胞免疫受容体DCIRの発現・精製およびX線結晶構造解析
福原秀雄, 松原永季, 神田諒, 矢部力朗, 海部知則, 尾瀬農之, 岩倉洋一郎, 前仲勝実, 日本蛋白質科学会年会プログラム・要旨集, 13th, 119, 31 May 2013
Japanese

パラミクソウイルス侵入機構と阻害剤開発
東端将哲, 福原秀雄, 齊藤貴士, 陳甦ぜい, 逢坂文那, 児玉耕太, 尾瀬農之, 前仲勝実, 日本薬学会年会要旨集(CD-ROM), 133年会, 1, 100, 100, Mar. 2013
(公社)日本薬学会, Japanese

結晶構造解析に向けた,乳がん特異的キナーゼBrkとアダプタータンパク質STAP‐2の機能解析
神田諒, 関根勇一, 前仲勝実, 松田正, 尾瀬農之, 日本分子生物学会年会プログラム・要旨集(Web), 36th, 2P-0302 (WEB ONLY), 2013
Japanese

麻疹ウイルス及び犬ジステンパーウイルスH蛋白質の構造学的研究
尾瀬農之, 橋口隆生, 酒匂幸, 伊藤由梨, 福原秀雄, 黒木喜美子, 柳雄介, 竹田誠, 前仲勝実, 日本蛋白質科学会年会プログラム・要旨集, 12th, 83, 31 May 2012
Japanese

機能および構造解析に向けた細胞表面蛋白質の調製法
福原秀雄, 橋口隆生, 黒木喜美子, 尾瀬農之, 前仲勝実, 日本蛋白質科学会年会プログラム・要旨集, 12th, 19, 31 May 2012
Japanese

ヤツメウナギ由来可変性リンパ球受容体VLRCの結晶構造
神田諒, 須藤洋一, 笠松純, 笠原正典, 前仲勝実, 尾瀬農之, 日本蛋白質科学会年会プログラム・要旨集, 12th, 117, 31 May 2012
Japanese

ロバスト設計最適化支援ツールを用いたin silicoスクリーニング
児玉耕太, 山口政隆, 小川修一, 尾瀬農之, 前仲勝実, 日本薬学会年会要旨集, 132nd, 4, 115, 05 Mar. 2012
Japanese

ヒト免疫制御受容体LILRB1とリガンドHLA‐Gの複合体の結晶構造解析
黒木喜美子, 松原永季, 神田諒, 上敷領淳, 尾瀬農之, 福永裕子, 前仲勝実, 日本分子生物学会年会プログラム・要旨集(Web), 35th, 1P-0060 (WEB ONLY), 2012
Japanese

樹状細胞免疫受容体DCIRの結晶化と構造解析
福原秀雄, 松原永季, 神田諒, 矢部力朗, 海部知則, 上敷領淳, 尾瀬農之, 岩倉洋一郎, 前仲勝実, 日本分子生物学会年会プログラム・要旨集(Web), 35th, 1P-0056 (WEB ONLY), 2012
Japanese

SrcおよびAuroraキナーゼ2重阻害による滑膜肉腫の相乗的in vivo抗腫瘍効果(Dual inhibition of Src and Aurora kinases abrogates tumor growth, invasion, and angiogenesis of synovial sarcoma in vivo)　　　　　　　　　　　　　　　
津田 真寿美, 新井 隆太, 渡部 琢哉, 尾瀬 農之, 小布施 力史, 前仲 勝実, 三浪 明男, 大場 雄介, 日本癌学会総会記事, 70回, 274, 274, Sep. 2011
日本癌学会, English

多様な形態を有するHLA‐G蛋白質とヒト免疫制御受容体との分子認識
黒木喜美子, 福永裕子, 上敷領淳, 白石充典, 尾瀬農之, 前仲勝実, 日本薬学会年会要旨集, 131年会, 1, 188, 188, Mar. 2011
(公社)日本薬学会, Japanese

イネいもち病菌の非病原性タンパク質AVR-Piaの分子間相互作用の解析
佐藤佑樹, 三木慎介, 尾瀬農之, 奥山雄大, 神崎洋之, 寺内良平, 曾根輝雄, 日本植物病理学会大会プログラム・講演要旨予稿集, 2011, 2011

SU6656 suppresses human synovial sarcoma progression through Src and Aurora kinase inhibition
Arai Ryuta, Tsuda Masumi, Watanabe Takuya, Ose Toyoyuki, Obuse Chikashi, Maenaka Katsumi, Minami Akio, Ohba Yusuke, 日本分子生物学会年会プログラム・要旨集(Web), 34th, WEB ONLY 2P-0690, 2011
Summary national conference

ヤツメウナギ由来可変性リンパ球受容体の結晶学的研究
神田諒, 須藤洋一, 笠松純, 笠原正典, 前仲勝実, 尾瀬農之, 日本結晶学会年会講演要旨集, 2011, 90, 2011
Japanese

麻疹ウイルスH蛋白質と受容体の複合体構造
尾瀬農之, 橋口隆生, 久保田万理恵, 真板宣夫, 柳雄介, 前仲勝実, 日本結晶学会年会講演要旨集, 2011, 87, 2011
Japanese

CDV‐Hの結晶構造解析と機能解析
伊藤由梨, 福原秀雄, 酒匂幸, 橋口隆生, 梶川瑞穂, 竹田誠, 柳雄介, 尾瀬農之, 前仲勝実, 日本結晶学会年会講演要旨集, 2011, 90, 2011
Japanese

ヒト免疫制御受容体LILRB1とリガンドHLA‐Gの複合体の結晶構造解析
松原永季, 黒木喜美子, 上敷領淳, 尾瀬農之, 前仲勝実, 日本結晶学会年会講演要旨集, 2011, 95, 2011
Japanese

HIV由来変異ペプチドによるKIR2DL1を介した免疫逃避機構の分子基盤
黒木喜美子, 上敷領淳, 尾瀬農之, THANANCHAI Hathairat, MAKADZANGE Tariro, ROWLAND‐JONES Sarah, DONG Tao, 前仲勝実, 日本結晶学会年会講演要旨集, 2011, 94, 2011
Japanese

HSV‐1のエンベロープ蛋白質gBペプチドおよびペア型レセプターPILRαの複合体結晶構造解析
尾瀬農之, 山口宗親, WAN Jing, 黒木喜美子, 田畑栄一, 真板宣夫, 中村聖子, 梶川瑞穂, 白鳥行大, 佐藤毅史, 荒瀬尚, 前仲勝実, 日本ウイルス学会学術集会プログラム・抄録集, 58th, 420, 15 Oct. 2010
Japanese

クラスI認識受容体の構造とリガンド認識
黒木喜美子, 福永裕子, 上敷領淳, 白石充典, 尾瀬農之, 前仲勝実, MHC, 17, 2, 136, 136, Aug. 2010
日本組織適合性学会, Japanese

ヒトのセレノシステイン導入系におけるL30の役割
川口亜希子, 尾瀬農之, 中村彰良, 喜多俊介, 姚閔, 田中勲, 日本蛋白質科学会年会プログラム・要旨集, 10th, 110, 15 May 2010
Japanese

大腸菌23S rRNAメチル基転移酵素YcbYのRNA結合能
喜多俊介, 木村聡, 尾瀬農之, 鈴木勉, 姚閔, 田中勲, 日本蛋白質科学会年会プログラム・要旨集, 10th, 128, 15 May 2010
Japanese

麻疹ウイルスによる細胞侵入機構の構造基盤
橋口隆生, 尾瀬農之, 上敷領淳, 柳雄介, 前仲勝実, 日本蛋白質科学会年会プログラム・要旨集, 10th, 32, 15 May 2010
Japanese

細胞表面において生体防御に関わる分子認識の構造生物学
福原秀雄, 橋口隆生, 黒木喜美子, 白石充典, 佐々木香織, 梶川瑞穂, 尾瀬農之, 前仲勝実, 日本蛋白質科学会年会プログラム・要旨集, 10th, 32, 15 May 2010
Japanese

酢酸菌由来CesDとCcpAxの相互作用解析
藤原孝彰, 尾瀬農之, 姚閔, 田島健次, 田中勲, 日本蛋白質科学会年会プログラム・要旨集, 10th, 144, 15 May 2010
Japanese

ペア型レセプターPILRα及びHSV‐1のエンベロープ蛋白質gB模倣ペプチド複合体結晶構造
尾瀬農之, 山口宗親, 黒木喜美子, 田畑栄一, 真板宣夫, 梶川瑞穂, 中村聖子, WANG Jing, 佐藤毅史, 荒瀬尚, 前仲勝実, 日本結晶学会年会講演要旨集, 2010, 97, 2010
Japanese

麻疹ウイルスHタンパク質と受容体SLAMの複合体の結晶構造と麻疹ウイルスの細胞侵入機構
橋口隆生, 尾瀬農之, 上敷領淳, 竹田誠, 前仲勝実, 柳雄介, 日本ウイルス学会学術集会プログラム・抄録集, 57th, 149, 01 Oct. 2009
Japanese

Paired Immunoglobulin(Ig)Like type 2 Receptor(PILR)αによるGlycoprotein B(gB)認識機構の解明
山口宗親, 黒木喜美子, 田畑栄一, 真板宣夫, 梶川瑞穂, 尾瀬農之, 中村聖子, 王静, 佐藤毅, 荒瀬尚, 前仲勝実, 日本ウイルス学会学術集会プログラム・抄録集, 57th, 149, 01 Oct. 2009
Japanese

真正細菌におけるセレノシステイン特異的翻訳伸長因子SelBとSECIS mRNAの相互作用基盤
尾瀬農之, RASUBALA Linda, 神田大輔, SOLER Nicolas, 吉澤聡子, FOURMY Dominique, 前仲勝実, 生化学, ROMBUNNO.2S8A-2, 25 Sep. 2009
Japanese

酢酸菌由来セルロース合成関連タンパク質の調製
藤原孝彰, 尾瀬農之, 田島健次, 田中勲, YAO Min, セルロース学会年次大会講演要旨集, 16th, 151, 152, 20 Jun. 2009
Japanese

Pyrococcus horikoshii由来アラニルtRNA合成酵素の結晶構造
徳永啓太, 曽我部正彰, 尾瀬農之, 中村彰良, 濡木理, 姚閔, 田中勲, PFシンポジウム要旨集, 26th, 2009

アラニルtRNA合成酵素の結晶構造より提唱されたtRNA再結合モデル
徳永啓太, 曽我部正彰, 尾瀬農之, 中村彰良, 濡木理, 姚閔, 田中勲, 日本蛋白質科学会年会プログラム・要旨集, 9th, 2009

マウスMHCクラスIb分子MILL2の結晶構造解析
梶川瑞穂, 尾瀬農之, 笠原正典, 前仲勝実, 生化学, 81回・31回, 2T20-1, 1, Nov. 2008
(公社)日本生化学会, Japanese

“21番目のアミノ酸”セレノシステインを取り込むための分子基盤
尾瀬農之, RASUBALA Linda, 神田大輔, SOLER Nicolas, 吉澤聡子, FOURMY Dominique, 前仲勝実, 補体シンポジウム講演集, 45th, 194, 194, Jul. 2008
(一社)日本補体学会, Japanese

複数の結合様式を介したミトコンドリアTom20によるプレ配列の認識機構
齊藤貴士, 井倉真由美, 尾瀬農之, 前仲勝実, 神田大輔, 日本蛋白質科学会年会プログラム・要旨集, 8th, 19, 23 May 2008
Japanese

構造基盤解析に向けたミトコンドリアTom20‐プレ配列ペプチド複合体の安定化
齊藤貴士, 井倉真由美, 尾瀬農之, 帯田孝之, 小島理恵子, 前仲勝実, 神田大輔, Abstr Annu Meet NMR Soc Jpn, 46th, 164, 165, 11 Sep. 2007
Japanese

PriAタンパク質によるDNA3′末端の塩基非選択的認識機構の解明
佐々木香織, 尾瀬農之, 岡本直明, 田中卓, 前仲勝実, 正井久雄, 斉藤美保子, 白井剛, 神田大輔, 日本蛋白質科学会年会プログラム・要旨集, 7th, 50, 07 May 2007
Japanese

ミトコンドリアTom20によるプレ配列の動的認識機構:緩和解析からのアプローチ
齊藤貴士, 井倉真由美, 尾瀬農之, 帯田孝之, 小島理恵子, 前仲勝実, 神田大輔, 日本蛋白質科学会年会プログラム・要旨集, 7th, 38, 07 May 2007
Japanese

Molecular basis for LILR recognition of HLA-G and its dimer
M. Shiroishi, K. Kuroki, T. Ose, L. Rasubala, I. Shiratori, H. Arase, D. Kohda, K. Maenaka, TISSUE ANTIGENS, 68, 4, CP6, CP6, Oct. 2006
BLACKWELL PUBLISHING, English, Summary international conference

Molecular Basis for the Selenocysteine Incorporation
OSE Toyoyuki, RASUBALA Linda, YOSHIZAWA Satoko, FOURMY Dominique, KOHDA Daisuke, MAENAKA Katsumi, Biophysics, 46, 2, 102, 105, 25 Mar. 2006
日本生物物理学会, Japanese

Crystallization and preliminary crystallographic analysis of the N-terminal domain of PriA from Escherichia coli
K Sasaki, T Ose, T Tanaka, T Mizukoshi, T Ishigaki, K Maenaka, H Masai, D Kohda, BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, 1764, 1, 157, 160, Jan. 2006
PriA, a DEXH-type DNA helicase, binds specifically to the 3' end of DNA through its N-terminal domain, and is a candidate sensor protein that recognizes arrested DNA replication forks in bacteria. We crystallized an N-terminal fragment of PriA in the absence and the presence of oligonucleotides to elucidate the structural basis for the specific recognition of the 3' terminus of DNA. (c) 2005 Elsevier B.V. All rights reserved., ELSEVIER SCIENCE BV, English

ミトコンドリアプレ配列認識のためのTom20タンパク質の動的構造
井倉真由美, 帯田孝之, 尾瀬農之, 遠藤斗志也, 前仲勝実, 神田大輔, 日本分子生物学会年会講演要旨集, 28th, 602, 25 Nov. 2005
Japanese

E.coli由来PriAのN末端ドメインによるDNA3′末端認識の構造的基盤
佐々木香織, 尾瀬農之, 田中卓, 岡本直明, 前仲勝実, 正井久雄, 神田大輔, 日本分子生物学会年会講演要旨集, 28th, 393, 25 Nov. 2005
Japanese

21番目のアミノ酸セレノシステインを取り込むための分子基盤
尾瀬 農之, Rasubala Linda, 神田 大輔, 構造生物, 11, 1, 21, 30, 2005
国際科学振興財団構造生物学坂部プロジェクト, Japanese

大腸菌由来PriAとオリゴヌクレオチドとの複合体の結晶化
佐々木香織, 尾瀬農之, 岡本直明, 田中卓, 正井久雄, 前仲勝実, 神田大輔, 日本分子生物学会年会プログラム・講演要旨集, 27th, 618, 25 Nov. 2004
Japanese

ラットTom20タンパク質とALDHプレ配列複合体のX線結晶構造解析
井倉真由美, 帯田孝之, 尾瀬農之, 遠藤斗志也, 前仲勝実, 神田大輔, 日本分子生物学会年会プログラム・講演要旨集, 27th, 732, 25 Nov. 2004
Japanese

ミトコンドリア・プレ配列受容体Tom20‐ALDHプレ配列複合体の結晶構造と緩和解析
帯田孝之, 井倉真由美, 尾瀬農之, 前仲勝実, 遠藤斗志也, 神田大輔, NMR討論会講演要旨集, 43rd, 212, 213, 10 Nov. 2004
Japanese

大腸菌yicl産物の機能およびX線構造解析
奥山正幸, 尾瀬農之, 北村百世, 森春英, 千葉誠哉, 木村淳夫, 田中勲, J Appl Glycosci, 51, Suppl., 40, 20 Jul. 2004
Japanese

Crystal Structure Analysis of Natural Diels-Alderase
OSE Toyoyuki, YAO Min, OIKAWA Hideaki, TANAKA Isao, Nihon Kessho Gakkaishi, 45, 6, 384, 390, 25 Dec. 2003
The crystal structure of natural Diels-Alderase, macrophomate synthase, in complex with pyruvate was determined at 1.7 Å resolution. The structure explains broad substrate specificity and provides mechanistic information regarding the multiple catalyst of this enzyme. The active site reveals several ingenious features for catalyzing the Diels-Alder reaction, which include the existence of residues for making catalytically important hydrogen bonds to the 2-pyrone. The rather large and hydrophobic milieu in the active site cavity allows a large-scale structural reorganization of the product i..., The Crystallographic Society of Japan, Japanese

41 Intermolecular Diels-Alderase, Macrophomate Synthase : Proposal for Reaction Mechanism Based on the Structure of the Active Site
Oikawa Hideaki, Watanabe Kenji, Mie Takashi, Ichihara Akitami, Honma Mamoru, Ose Toyoyuki, Yao Min, Tanaka Isao, Symposium on the Chemistry of Natural Products, symposium papers, 44, 241, 246, 01 Sep. 2002
Macrophomate synthase catalyzes unusual multistep reaction from oxalacetate and 2-pyrone to fungal metabolite, macrophomate involving Diels-Alder reaction. To elucidate its reaction mechanism, requirement of metal ion, kinetic parameter and stereochemical course were investigated. In addition, the structure of the active site of macrophomate synthase was determined by X-ray crystallography., Symposium on the chemistry of natural products, Japanese

植物ホルモンの生合成を阻害する蛋白質の立体構造解析
尾瀬 農之, 姚 閔, 田中 勲, 杉山産業化学研究所年報, 1999, 73, 87, 1999
杉山産業化学研究所, Japanese

量子生命科学ハンドブック
尾瀬農之,小林正人, 第3節 水素原子核の量子効果がもたらす特徴的な生体内化学反応とその量子化学計算技術
エヌ・ティー・エス, Mar. 2024, 9784860438821, iii, x, 346, viiip, Japanese, [Contributor]

出力多様性を司るSTAT多量体化の構造基盤と，それに抗するウイルス蛋白質の解析
科学研究費助成事業
01 Apr. 2024 - 31 Mar. 2028
尾瀬 農之, 久米田 博之, 杉田 征彦, 姚 閔
日本学術振興会, 基盤研究(B), 北海道大学, 24K01959

Studies on molecular mechanism of effector-mediated establishment of host specificity in plant pathogens and their application
Grants-in-Aid for Scientific Research
05 Jul. 2021 - 31 Mar. 2026
高野 義孝, 大木 進野, 尾瀬 農之
本研究では、宿主特異性成立に関与する植物病原菌エフェクター（EPC1、EPC2、EPC3）の解析を起点に、エフェクターを介した植物病原菌の宿主特異性成立の分子基盤を解明することを目的とする。本年度は、まずエフェクターEPC1、EPC2、EPC3をベンサミアナタバコ葉に一過的に発現させ、その発現が同植物のPAMP誘導免疫に与える影響を調べた。その結果、EPC1、EPC2、EPC3いずれも、細菌のPAMP（flg22）が引き起こす活性酸素生成を抑制することを明らかにした。続いて、エピトープタグを付加した各EPCエフェクターをベンサミアナタバコ葉およびメロン子葉に一過的に発現させ、続いて免疫沈降解析を実施し、共沈降してくるタンパク質について、LC-MS/MS解析を実施し、標的因子候補のリスト化を完了した。また、EPC3の特定の領域について、その領域のみで機能が保持されることを明らかにし、さらにその領域を発現させた場合は、発現タンパク質が可溶化することを見出している。ウリ科作物への宿主特異性に関わるエフェクターを網羅的に同定するために、ウリ類炭疽病菌とウリ類炭疽病菌が属するオービクラクレード内の別種に対する比較オミクス解析を実施し、ウリ類炭疽病菌の分離株すべてにおいて存在し、他種では必ずしも存在しない遺伝子群、あるいは、ウリ類炭疽病菌において発現するが他種では必ずしも発現しない遺伝子群の選抜を完了した。さらに最近になり、EPC4と命名した新規のエフェクターの発見に成功している。EPC4は、EPC1、EPC2、EPC3と同様にウリ科作物への病原性には必要である一方、ベンサミアナタバコへの病原性には必須ではない。EPC4について、その一過的発現解析を実施した結果、EPC4がflg22が誘導する活性酸素生成を抑制することを見出している。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (S), Kyoto University, 21H05032

世界初IDP型酵素の存在意義と構造形成機構の解明
科学研究費助成事業
30 Jun. 2022 - 31 Mar. 2024
久米田 博之, 姚 閔, 尾瀬 農之
立体構造を形成しないIDPとして特に研究が進んでいる蛋白質は，高等生物シグナル伝達系や転写翻訳系に多く含まれ，他の蛋白質との相互作用時に過渡的部分構造形成をおこなうものも多い。一方で，ポリペプチド鎖の大部分がIDPで構成される酵素はこれまで報告が無い。天然に存在するポリエーテル化合物は，多様かつ強力な生物活性を示し，産業応用や細胞生物学への利用が多く見られる。これらのポリエーテル化合物は、テトラヒドロフラン環やテトラヒドロピラン環が多くの不斉点を介し連結されており，構造多様性はエーテル環の数や順序，大きさが決定している。ポリエーテル骨格の構築機構は，鎖状ポリオレフィン前駆体がエポキシ化され，引き続き位置選択的なエポキシド開環反応によりポリエーテル骨格が連鎖的に構築される。私達は，monensin生合成をモデルケースとしてポリエーテル骨格構築機構の解明に取り組んできた。Monensinの場合，その骨格を構築するために3 回の5-exo 酵素環化反応が必要である。これまでポリエーテル天然物生合成経路に存在するエポキシド開環エーテル環化酵素はIDP型の酵素であることを提唱し，構造形成機構や存在意義の解析を進めてきた。結晶構造解析，1H-15N HSQC (NMR), CD, DSC, Native-MSを組み合せ，モネンシン生合成経路中に存在する蛋白質のうち，１つは酵素活性を持たず，もう一方の構造形成を補助するシャペロン様分子であることを示すことができた。一般的にポリエーテル環化反応進行のためには，長大な基質を収納するポケットが必要であり，また，環化反応をうける基質のエポキシドおよび水酸基周辺の構造変化に対応できる柔軟な活性部位が必要である。我々のペア型酵素仮説を使用すると，酵素がこういった課題を解決するために採用している戦略を説明できる。
日本学術振興会, 挑戦的研究(萌芽), 北海道大学, 22K19282

Molecular basis of counteraction against the JAK-STAT signaling pathway by virus
Grants-in-Aid for Scientific Research
01 Apr. 2021 - 31 Mar. 2024
尾瀬 農之, 久米田 博之, 杉田 征彦, 于 健
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 21H02408

新規に開発したＢＬＬナノ構造体による、タンパク質結晶核形成過程の研究
科学研究費助成事業
01 Apr. 2021 - 31 Mar. 2024
姚 閔, 尾瀬 農之
日本学術振興会, 基盤研究(B), 北海道大学, 21H01754

Antibodies targeting dynamic epitopes generated by posttranslational protein glycosylation
Grants-in-Aid for Scientific Research
01 Apr. 2019 - 31 Mar. 2023
西村 紳一郎, 田中 良和, 比能 洋, 尾瀬 農之
本研究課題は、申請者らの独創的な「動的エピトープを標的とする新薬開発」という発想と、既にその基盤が完成しており効率的でシームレスな抗体開発までを可能とする「糖鎖工学プラットフォームを活用する戦略」によって創出されたED抗体（epitope-defined antibody）を用いて、主としてがん領域におけるアンメットメディカルニーズに応え得る新たながん治療薬の開発を目的としている。
この研究課題においては多くのがん細胞表面に高発現してがんの増殖や転移の促進などに深く関与することが広く知られるムチンMUC1の細胞外タンデムリピートドメインに生成する多様な動的エピトープ（dynamic epitope）を特異的な標的とするED抗体である抗MUC1モノクローナル抗体の構造改変と低分子化によって高機能化することで新たな創薬モダリティーを創出することを具体的な目標として進めている。
2021年度（３年目）はMUC1-STを動的エピトープとするSN131のFab-MUC1-ST共結晶の構造解析に成功した。また、物理化学的・生化学的キャラクタリゼーションに加えて、すい臓がん細胞表面のMUC1との結合やSiglecなどとの相互作用などを多角的に解析した（論文投稿準備中）。SN131の改変型抗体であるSN132（動的エピトープとの結合領域を含むanti-parallel coiled-coil構造のFv-claspとリン脂質DPC膜結合性membrane-associated peptide領域を融合した分子量40K程度の全く新しいタイプの機能性抗体フラグメント）の作成法を確立した（特許出願準備中）。
また、SN131およびSN121のFv領域を、2種類のリンパ球を認識する抗体（OKT3およびL2K）のFv領域と融合させて新たに８種の二重特異性抗体の作成を進めており高活性を示す高性能な二重特異性抗体取得に向けて研究が進展している。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (A), Hokkaido University, 19H00918

生体分子中の窒素の特性を，中性子回折と計算化学により解明する　　　　　　　　　　　　　　　
Grants-in-Aid for Scientific Research Fund for the Promotion of Joint International Research (Fostering Joint International Research (B))
07 Feb. 2019 - 31 Mar. 2022
尾瀬 農之, 齋藤 徹, 塚本 卓
核酸やアミノ酸等の窒素化合物を代謝する過程においては，アミンのプロトタイプであるアンモニアが発生する。その反応性のために様々な細胞毒性を引き起こすアンモニアは，代謝や輸送をおこなう上で体内で注意して取り扱われなければならない。血液中に誤って放出されたアンモニアは，脳血液関門を容易に通過し脳障害をも引き起こす。陸上動物はふんだんに水を使用できないため，爬虫類・鳥類では尿酸として，両生類・哺乳類では尿素としてアンモニアを排出する系を備えている。これら排出回路を含め，アンモニアを扱う酵素には特別な設計がなされているおり，これの破綻は病気につながる。例えば，カルバモイルリン酸合成酵素(CPS)，シチジン３リン酸合成酵素(CTS)，グルタミン依存型アミドトランスフェラーゼ(Gat)を始めとし，アンモニアを反応中間体として利用する酵素は，求核性の高いアミンであるアンモニアが外部（細胞質）に漏れないよう，活性部位間がトンネルで結ばれている。トンネル内でアンモニアがどのように輸送されているかは，非常に興味深いトピックである。疎水性トンネルではアンモニアのまま輸送されていると予測される。また，親水性トンネルではプロトン化されていると予想されるが，アミンの性質を使用し，プロトン化と脱プロトン化を繰り返しながら輸送されているかもしれない。いずれにしても，Ｘ線結晶解析では水分子とアンモニア分子の違いを見分けることができないため，実験科学はデッドエンドになっていた。本提案では中性子線回折の利用方法をさらに進歩させ，窒素原子の持つ特徴を活かした構造生物学へと拡張する。アンモニアの分子内トンネルを使用した輸送機構は古細菌，真正細菌，真核生物に普遍的に見られる現象であるが，分子メカニズムの詳細は謎のままである。とりわけ，輸送に必要な水との協調作用を知る上では水分子と明確に区別する必要がある。
Japan Society for the Promotion of Science, Fund for the Promotion of Joint International Research (Fostering Joint International Research (B)), Hokkaido University, Principal investigator, Competitive research funding, 18KK0194

生体分子中におけるアミンの量子特性を解明する　　　　　　　　　　　　　　　
さきがけ
Oct. 2018 - Mar. 2022
尾瀬農之
JST, Principal investigator, Competitive research funding

必要時に可逆的立体構造形成する新規ペア型エーテル環化酵素の解析と再設計による応用　　　　　　　　　　　　　　　
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
01 Apr. 2019 - 31 Mar. 2021
尾瀬 農之
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), Hokkaido University, 19H04634

Elucidation of a tRNA conversion mechanism of transsulfursome by the structural analysis of quaternary complex
Grants-in-Aid for Scientific Research
01 Apr. 2017 - 31 Mar. 2020
Yao Min
To translate the protein according to the genetic code on the ribosome, the aminoacyl-tRNA (aa-tRNA) must be synthesized correctly. Generally, the aminoacyl-tRNA synthetase (aaRS) directly catalyzes this synthesis. However, in methanogenic archaea, a ternary complex called transsulfursome (SepRS, SepCys, SepCysS) synthesizes Cys-tRNA(Cys) in an indirect pathway.
In this study, we analyzed the relationship of structure and function of transsulfursome-tRNA complex by using X-ray crystallography, small-angle X-ray scattering, electron microscopic observation, and biochemical methods. Taken results together, we understood the dynamic molecular mechanism for the Cys-tRNA(Cys) synthesis of the transsulfursome.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 17H03637

JAK-STAT経路を不活化するため，ウイルスが採用する様々な戦略の分子機構解析　　　　　　　　　　　　　　　
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
01 Apr. 2017 - 31 Mar. 2020
尾瀬 農之
今回私達は, ITC測定を用いてMeV-VがSTAT1よりSTAT2に対して特異性が高いことを明らかにした。そこで，STAT2を主な標的としてType I IFNシグナル経路を阻害していると考えた。STAT2は, Type I IFNシグナル経路において, リン酸化, ヘテロ複合体化, 核移行, 転写活性化という過程を経る。この過程の中でMeV-VがSTAT2に干渉してシグナル伝達を阻害することを考えると, メカニズムとして リン酸化阻害， ヘテロ複合体化阻害，核移行阻害，転写活性化阻害が考えられた。
リン酸化阻害について, STAT2のリン酸化前後でMeV-Vの結合親和性が変化するか検証することを考え, 大腸菌TKB1でSTAT2を調製することを試みた。しかし, STAT1と同じ精製法でSTAT2を精製することはできなかった。また, リン酸化させたSTAT2は非常に凝集しやすい性質であった。バッファーや精製条件の検討により, リン酸化STAT2の調製を試みたいと考えている。したがってMeV-VのSTAT2に対する結合がリン酸化阻害に関わっているか不明であるが, Type I IFN経路阻害メカニズムとして現在多くの研究者に受け入れられているのは, MeV-VによるSTAT1やSTAT2のリン酸化阻害である（Chambers R. et al., 2009; Audsley M. D. et al., 2013）。私達が組換え調製したpY-STAT1を使ったITCによる相互作用解析では, MeV-Vとの相互作用が観測できなかった。これは, 非リン酸化STAT1の時に露出していた相互作用面がリン酸化に伴うコンホメーション変化によって隠れてしまったものと考えられ, MeV-Vはリン酸化を受ける前のSTAT1に対して特異的に結合する。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 17K07296

JAK-STAT経路を不活化するため，ウイルスが採用する様々な戦略の分子機構解析　　　　　　　　　　　　　　　
Apr. 2017 - Mar. 2020
尾瀬農之
科学研究費補助金（基盤C）, Principal investigator, Competitive research funding

４者複合体の構造解析によるtranssulfursomeのｔＲＮＡ変換機構の解明　　　　　　　　　　　　　　　
Apr. 2017 - Mar. 2020
姚閔
科学研究費補助金（基盤B）, Competitive research funding

Molecular mechanism analysis of STAT activation in breast tumor cells regulated by adaptor protein
Grants-in-Aid for Scientific Research
01 Apr. 2014 - 31 Mar. 2017
OSE Toyoyuki
The non-receptor tyrosine kinase breast tumor kinase (Brk), has been identified as a highly expressed Protein-tyrosine kinases in human melanocyte. Brk was reported to play key roles for cancer progression via overactivation of Signal Transducers and Activator of Transcription (STAT), especially STAT3 and STAT5. This activation is mediated by an adaptor protein STAP-2, which expresses ubiquitously and harobors many important functions for cell signal regulation. We successfully expressed recombinant Brk and STAP-2 including their mutants, checked kinase activity, monitored interactions, and structurally analyzed using small angle x-ray scattering (SAXS). The C-terminal phosphorylation site of Brk is, on the contrary to our expectation, didn’t produce open/close conformational change.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 26440016

アダプタータンパク質が担う，乳がん細胞における転写因子STATの活性化基盤の解明　　　　　　　　　　　　　　　
Apr. 2014 - Mar. 2017
尾瀬農之
科学研究費補助金（基盤C）, Principal investigator, Competitive research funding

Molecular basis regulating multiple signals by an adaptor protein
Grants-in-Aid for Scientific Research
01 Apr. 2012 - 31 Mar. 2014
OSE TOYOYUKI
The non-receptor tyrosine kinase breast tumor kinase (Brk), also known as PTK6, has been identified as a highly expressed Protein-tyrosine kinases in human melanocyte.
STAP-2 is also known as breast tumor kinase (Brk) substrate (BKS). it is important to better understand the contribution of Brk kinase activity and protein interactions to the STAT3-mediated signal transduction pathways in breast cancer. Brk phosphorylates STAP-2 and phosphorylated STAP-2 is believed to play an important role in Brk-mediated STAT3 activation. We purified Brk, STAP-2, and STAT3 and checked the kinase activity in vitro. Using purified proteins, we also checked the binding affinity between Brk and STAP-2. The information obtained by small angle xray scattering (SAXS) experiments was also useful to judge whether the conformational change of Brk is included (open/closed).
Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (B), Hokkaido University, Principal investigator, Competitive research funding, 24770087

Elucidation of molecular mechanism of rice blast resistance
Grants-in-Aid for Scientific Research
01 Apr. 2011 - 31 Mar. 2014
SONE Teruo, TERAUCHI Ryohei, OSE Toyoyuki
Rice blast is the most important disease or rice. Blast is usually controlled by agrochemicals and resistant cultivars, but knowledge about molecular mechanisms of rice resistance toward blast is limited. This study aimed to elucidate the molecular mechanism of rice blast resistance by analyzing interaction of rice blast resistance gene Pia and blast virulence gene AVR-Pia. Results of this study indicated that AVR-Pia gene is started to be expressed before the penetration into rice cells, and after the secretion, AVR-Pia protein forms homodimer, and detected by rice Pia gene product. This study will contribute to further understanding of rice-rice blast interaction, which is the first step of resistance induction.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 23380024

Analysis of translation mechanism for selenoprotein synthesis
Grants-in-Aid for Scientific Research
2010 - 2011
OSE Toyoyuki
We successfully prepared protein and RNA molecules which are required for selecocystein incorporation on ribosome. These molecules are SBP2, L30, EFSec, SEICIS-RNA, tRNASec and subjected to biochemical and biophysical experiments. Crystal structure of L30 could be analyzed and published. Since we originally succeeded generating functional tRNASec, binding assay among tRNASec, EFSec, and SBP2 in the presence of specific amino acid are now in progress. We observed the significant binding affinity(K_d=229 nM) between the wild type and GDP using ITC, the way to prepare the molecule was thus confirmed. Furthermore, mutants generated for the purpose of crystallization were proved that they have proper secondary structures
Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (B), Hokkaido University, Principal investigator, Competitive research funding, 22770093

第3の可変的リンパ球レセプターの構造と機能:リンパ球サブセットの起源
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas
2010 - 2011
笠原 正典, 尾瀬 農之
獲得免疫系と、それを支えるリンパ球は長い間、有顎類に固有であると考えられてきた。ところが、leucine-rich repeatモジュールを組み換えることにより10^<10>を超える多様性を生み出す抗原レセプターである可変性リンパ球レセプター(variable lymphocyte receptor、以下VLR)が無顎類ヤツメウナギで発見されるに及んで、この考えは根底から覆されるに至った。先に、われわれは、VLRにAとBが存在することを示したが、最近、VLRAとBはそれぞれT、B様のリンパ球に発現され、Aは膜結合型レセプターとして、Bは抗体として機能することが示された。以上から、無顎類の段階で、細胞性免疫、液性免疫を担うリンパ球のサブセットがすでに存在することが明らかになった。今回、我々は第3のVLRを発見し、それをVLRCと命名した。本年度の研究により、1)VLRCはVLA+細胞、VLRB+細胞とは別のリンパ球サブセットに発現されていること、2)VLRCは分泌されないこと、3)VLRCは配列的には、VLRBに対してよりVLRAに類似性が高いこと、4)まれに単一リンパ球においてVLRAとVLRCの両者の組み換えが起きるが、一方のみが機能的なVLRタンパク質をコードできることなどが明らかになった。おそらく、VLRC+細胞はVLRA+細胞と同様、T細胞系の細胞と想定される。したがって、有顎類に3種のリンパ球サブセット(B、αβT、γδT細胞)が存在するように、無顎類にも3種のリンパ球サブセットが存在し、そのうちの二つはT細胞様で、一つはB細胞様であることが示唆された。研究分担者はVLRCの結晶を得て、その構造解析に成功した。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Priority Areas, Hokkaido University, Coinvestigator not use grants, Competitive research funding, 22021001

発熱現象を担う哺乳類の脱共役蛋白質の機能・構造解析
Grants-in-Aid for Scientific Research Grant-in-Aid for JSPS Fellows
2005 - 2005
尾瀬 農之
Japan Society for the Promotion of Science, Grant-in-Aid for JSPS Fellows, Kyushu University, 05J05880

エチレン前駆体ACCを分解する酵素反応の解析〜立体構造からのアプローチ〜
Grants-in-Aid for Scientific Research Grant-in-Aid for JSPS Fellows
2000 - 2002
尾瀬 農之
炭素-炭素結合を協奏的に形成する反応を触媒する酵素(macrophomate synthase)の立体構造解析に成功した。得られた立体構造から、このステップはDiels-Alder反応の可能性が極めて高くなった。Diels-Alder反応は、シクロヘキセンを合成する有機化学上極めて重要な反応である。この反応を触媒する酵素の立体構造は世界初のものであり、Nature誌で発表した。活性部位は巧妙に設計されており、より効率的にDiels-Alder反応を進行させるための戦略を見ることができる。基質を導入した活性部位の構造及び、変異実験や反応中間体の絶対配置に関する情報を組み合わせ、1)より活性化した基質を用いること、2)水素結合によって反応性を大きく上昇させること、3)生成物阻害を回避する構造変化を伴うこと、4)柔軟性に富んだループ領域をもっていることなどを具体的に明らかにした。この活性部位は、新たな化合物を作るための、抗体触媒を設計する上で非常に重要な情報となるものである。
三員環アミノ酸ACC(1-aminocyclopropane-1-carboxylate)を分解できる特殊な酵素ACC deaminase(yACCD;酵母Hansenula saturunus由来)の結晶構造を、分解能2.0Åで精密化した。立体構造および構造情報から予測した反応機構を、J.Biol.Chem.誌上に発表した。この反応機構を検証するために変異体を9種類作製した。活性消失が確認された変異体のうち、K51T, Y295FとACCを結合させた複合体の結晶化に成功し、反応中間体構造を得た。反応中でビタミンB6は基質と結合し、回転することで反応に最適な立体環境を提供する。活性残基Lys51はACCのメチレン側鎖からプロトンを引き抜くモデルを提唱することができた。これはビタミンB6酵素では初のものであり、進化の多様性を表すモデルと言える。論文投稿中である。また、超高度好熱古細菌P.horikoshiiにおいてACCDとアサインされた遺伝子(PH0054)がコードする蛋白質の、ACC存在下での構造解析にも成功し、大きなドメイン変化が観察できた。ACCD活性の発現は当初の予想以上に厳密な環境制御を要している。論文投稿準備中である。
Japan Society for the Promotion of Science, Grant-in-Aid for JSPS Fellows, Hokkaido University, 00J07161