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Miki Hara-Yokoyama, Mutsuko Kukimoto-Niino,
Kazue Terasawa,
Satoru Harumiya,
Katarzyna A Podyma-Inoue,
Nobumasa Hino,
Kensaku Sakamoto,
Satsuki Itoh,
Noritaka Hashii,
Yoko Hiruta, [......],
Motoaki Wakiyama,
Mikako Shirouzu,
Takeshi Kasama,
Hiroshi Takayanagi,
Naoko Utsunomiya-Tate,
Kiyoshi Takatsu,
Toshiaki Katada,
Yoshio Hirabayashi,
Shigeyuki Yokoyama,
Masaki Yanagishita
[show abstract]
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ABSTRACT: The leukocyte cell-surface antigen CD38 is the major nicotinamide adenide dinucleotide glycohydrolase in mammals, and its ectoenzyme activity is involved in calcium mobilization. CD38 is also a raft-dependent signaling molecule. CD38 forms a tetramer on the cell surface, but the structural basis and the functional significance of tetramerization have remained unexplored. We identified the interfaces contributing to the homophilic interaction of mouse CD38 by site-specific crosslinking on the cell surface with an expanded genetic code, based on a crystallographic analysis. A combination of the three interfaces enables CD38 to tetramerize: one interface involving the juxtamembrane α-helix is responsible for the formation of the core dimer, which is further dimerized via the other two interfaces. This dimerization of dimers is required for the catalytic activity and the localization of CD38 in membrane rafts. The glycosylation prevents further self-association of the tetramer. Accordingly, the tetrameric interaction underlies the multifaceted actions of CD38.
Structure 08/2012; 20(9):1585-95. · 6.35 Impact Factor
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Tomohiro Sato,
Hisami Watanabe,
Keiko Tsuganezawa,
Hitomi Yuki,
Junko Mikuni,
Seiko Yoshikawa, Mutsuko Kukimoto-Niino,
Takako Fujimoto,
Yumiko Terazawa,
Motoaki Wakiyama,
Hirotatsu Kojima,
Takayoshi Okabe,
Tetsuo Nagano,
Mikako Shirouzu,
Shigeyuki Yokoyama,
Akiko Tanaka,
Teruki Honma
[show abstract]
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ABSTRACT: EGFR is a target protein for the treatment of non small cell lung cancer (NSCLC). The mutations associated with the activation of EGFR kinase activity, such as L858R and G719S, destabilize the inactive conformation of EGFR and are closely linked with the development of NSCLC. The additional T790M mutation reportedly causes drug resistance against the commercially available EGFR inhibitors, gefitinib and erlotinib. In this study, we searched for novel G719S/T790M EGFR inhibitors by a new in silico screening strategy, using two datasets. The results of in silico screening using protein-ligand docking are affected by the selection of 3D structure of the target protein. As the first strategy, we chose the 3D structures for in silico screening by test dockings using the G719S/T790M crystal structure, its molecular dynamics snapshots, and known inhibitors of the drug-resistant EGFR. In the second strategy, we selected the 3D structures by test dockings using all of the EGFR structures, regardless of the mutations, and all of the known EGFR inhibitors. Using each of the 3D structures selected by the strategies, 1000 compounds were chosen from the 71,588 compounds. Kinase assays identified 15 G719S/T790M EGFR inhibitors, including two compounds with novel scaffolds. Analyses of their structure-activity relationships revealed that interactions with the mutated Met790 residue specifically increase the inhibitory activity against G719S/T790M EGFR.
Bioorganic & medicinal chemistry 04/2012; 20(12):3756-67. · 2.82 Impact Factor
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Akihiko Nishikimi,
Takehito Uruno,
Xuefeng Duan,
Qinhong Cao,
Yuji Okamura,
Takashi Saitoh,
Nae Saito,
Shunsuke Sakaoka,
Yao Du,
Atsushi Suenaga, [......],
Takayoshi Okabe,
Fumiyuki Sanematsu,
Yoshihiko Tanaka,
Hideki Sumimoto,
Teruki Honma,
Shigeyuki Yokoyama,
Tetsuo Nagano,
Daisuke Kohda,
Motomu Kanai,
Yoshinori Fukui
[show abstract]
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ABSTRACT: Tissue infiltration of activated lymphocytes is a hallmark of transplant rejection and organ-specific autoimmune diseases. Migration and activation of lymphocytes depend on DOCK2, an atypical Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 does not contain Dbl homology domain typically found in guanine nucleotide exchange factors, DOCK2 mediates the GTP-GDP exchange reaction for Rac through its DHR-2 domain. Here, we have identified 4-[3'-(2″-chlorophenyl)-2'-propen-1'-ylidene]-1-phenyl-3,5-pyrazolidinedione (CPYPP) as a small-molecule inhibitor of DOCK2. CPYPP bound to DOCK2 DHR-2 domain in a reversible manner and inhibited its catalytic activity in vitro. When lymphocytes were treated with CPYPP, both chemokine receptor- and antigen receptor-mediated Rac activation were blocked, resulting in marked reduction of chemotactic response and T cell activation. These results provide a rational of and a chemical scaffold for development of the DOCK2-targeting immunosuppressant.
Chemistry & biology 04/2012; 19(4):488-97. · 6.52 Impact Factor
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Seisuke Kusano, Mutsuko Kukimoto-Niino,
Nobumasa Hino,
Noboru Ohsawa,
Masashi Ikutani,
Satoshi Takaki,
Kensaku Sakamoto,
Miki Hara-Yokoyama,
Mikako Shirouzu,
Kiyoshi Takatsu,
Shigeyuki Yokoyama
[show abstract]
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ABSTRACT: Interleukin-5 (IL-5), a major hematopoietin, stimulates eosinophil proliferation, migration, and activation, which have been implicated in the pathogenesis of allergic inflammatory diseases, such as asthma. The specific IL-5 receptor (IL-5R) consists of the IL-5 receptor α subunit (IL-5RA) and the common receptor β subunit (βc). IL-5 binding to IL-5R on target cells induces rapid tyrosine phosphorylation and activation of various cellular proteins, including JAK1/JAK2 and STAT1/STAT5. Here, we report the crystal structure of dimeric IL-5 in complex with the IL-5RA extracellular domains. The structure revealed that IL-5RA sandwiches the IL-5 homodimer by three tandem domains, arranged in a "wrench-like" architecture. This association mode was confirmed for human cells expressing IL-5 and the full-length IL-5RA by applying expanded genetic code technology: protein photo-cross-linking experiments revealed that the two proteins interact with each other in vivo in the same manner as that in the crystal structure. Furthermore, a comparison with the previously reported, partial GM-CSF•GM-CSFRA•βc structure enabled us to propose complete structural models for the IL-5 and GM-CSF receptor complexes, and to identify the residues conferring the cytokine-specificities of IL-5RA and GM-CSFRA.
Protein Science 03/2012; 21(6):850-64. · 2.80 Impact Factor
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Yosuke Harada,
Yoshihiko Tanaka,
Masao Terasawa,
Markus Pieczyk,
Katsuyoshi Habiro,
Tomoya Katakai,
Kyoko Hanawa-Suetsugu, Mutsuko Kukimoto-Niino,
Tomoko Nishizaki,
Mikako Shirouzu,
Xuefeng Duan,
Takehito Uruno,
Akihiko Nishikimi,
Fumiyuki Sanematsu,
Shigeyuki Yokoyama,
Jens V Stein,
Tatsuo Kinashi,
Yoshinori Fukui
[show abstract]
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ABSTRACT: To migrate efficiently through the interstitium, dendritic cells (DCs) constantly adapt their shape to the given structure of the extracellular matrix and follow the path of least resistance. It is known that this amoeboid migration of DCs requires Cdc42, yet the upstream regulators critical for localization and activation of Cdc42 remain to be determined. Mutations of DOCK8, a member of the atypical guanine nucleotide exchange factor family, causes combined immunodeficiency in humans. In the present study, we show that DOCK8 is a Cdc42-specific guanine nucleotide exchange factor that is critical for interstitial DC migration. By generating the knockout mice, we found that in the absence of DOCK8, DCs failed to accumulate in the lymph node parenchyma for T-cell priming. Although DOCK8-deficient DCs migrated normally on 2-dimensional surfaces, DOCK8 was required for DCs to crawl within 3-dimensional fibrillar networks and to transmigrate through the subcapsular sinus floor. This function of DOCK8 depended on the DHR-2 domain mediating Cdc42 activation. DOCK8 deficiency did not affect global Cdc42 activity. However, Cdc42 activation at the leading edge membrane was impaired in DOCK8-deficient DCs, resulting in a severe defect in amoeboid polarization and migration. Therefore, DOCK8 regulates interstitial DC migration by controlling Cdc42 activity spatially.
Blood 03/2012; 119(19):4451-61. · 9.90 Impact Factor
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Kyoko Hanawa-Suetsugu, Mutsuko Kukimoto-Niino,
Chiemi Mishima-Tsumagari,
Ryogo Akasaka,
Noboru Ohsawa,
Shun-ichi Sekine,
Takuhiro Ito,
Naoya Tochio,
Seizo Koshiba,
Takanori Kigawa,
Takaho Terada,
Mikako Shirouzu,
Akihiko Nishikimi,
Takehito Uruno,
Tomoya Katakai,
Tatsuo Kinashi,
Daisuke Kohda,
Yoshinori Fukui,
Shigeyuki Yokoyama
[show abstract]
[hide abstract]
ABSTRACT: DOCK2, a hematopoietic cell-specific, atypical guanine nucleotide exchange factor, controls lymphocyte migration through ras-related C3 botulinum toxin substrate (Rac) activation. Dedicator of cytokinesis 2-engulfment and cell motility protein 1 (DOCK2•ELMO1) complex formation is required for DOCK2-mediated Rac signaling. In this study, we identified the N-terminal 177-residue fragment and the C-terminal 196-residue fragment of human DOCK2 and ELMO1, respectively, as the mutual binding regions, and solved the crystal structure of their complex at 2.1-Å resolution. The C-terminal Pro-rich tail of ELMO1 winds around the Src-homology 3 domain of DOCK2, and an intermolecular five-helix bundle is formed. Overall, the entire regions of both DOCK2 and ELMO1 assemble to create a rigid structure, which is required for the DOCK2•ELMO1 binding, as revealed by mutagenesis. Intriguingly, the DOCK2•ELMO1 interface hydrophobically buries a residue which, when mutated, reportedly relieves DOCK180 from autoinhibition. We demonstrated that the ELMO-interacting region and the DOCK-homology region 2 guanine nucleotide exchange factor domain of DOCK2 associate with each other for the autoinhibition, and that the assembly with ELMO1 weakens the interaction, relieving DOCK2 from the autoinhibition. The interactions between the N- and C-terminal regions of ELMO1 reportedly cause its autoinhibition, and binding with a DOCK protein relieves the autoinhibition for ras homolog gene family, member G binding and membrane localization. In fact, the DOCK2•ELMO1 interface also buries the ELMO1 residues required for the autoinhibition within the hydrophobic core of the helix bundle. Therefore, the present complex structure reveals the structural basis by which DOCK2 and ELMO1 mutually relieve their autoinhibition for the activation of Rac1 for lymphocyte chemotaxis.
Proceedings of the National Academy of Sciences 02/2012; 109(9):3305-10. · 9.68 Impact Factor
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[show abstract]
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ABSTRACT: The migratory properties of lymphocytes depend on DOCK2, an atypical Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 does not contain the Dbl homology domain typically found in guanine nucleotide exchange factors (GEFs), DOCK2 mediates the GTP-GDP exchange reaction for Rac via its DOCK homology region (DHR)-2 (also known as CZH2 or Docker) domain. DOCK2 DHR-2 domain is composed of three lobes, and Rac binding site and catalytic center are generated entirely from lobes B and C. On the other hand, lobe A has been implicated in dimer formation, yet its physiological significance remains unknown. Here, we report that lobe A-mediated DOCK2 dimerization is crucial for Rac activation and lymphocyte migration. We found that unlike wild-type DOCK2, DOCK2 mutant lacking lobe A failed to restore motility and polarity when expressed in thymoma cells and primary T cells lacking endogenous expression of DOCK2. Similar results were obtained with the DOCK2 point mutant having a defect in dimerization. Deletion of lobe A from the DHR-2 domain did not affect Rac GEF activity in vitro. However, fluorescence resonance energy transfer analyses revealed that lobe A is required for DOCK2 to activate Rac effectively during cell migration. Our results thus indicate that DOCK2 dimerization is functionally important under the physiological condition where only limited amounts of DOCK2 and Rac are localized to the plasma membrane.
PLoS ONE 01/2012; 7(9):e46277. · 4.09 Impact Factor
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[show abstract]
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ABSTRACT: The calcitonin receptor-like receptor (CRLR), a class B GPCR, forms a heterodimer with receptor activity-modifying protein 2 (RAMP2), and serves as the adrenomedullin (AM) receptor to control neovascularization, while CRLR and RAMP1 form the calcitonin gene-related peptide (CGRP) receptor. Here, we report the crystal structures of the RAMP2 extracellular domain alone and in the complex with the CRLR extracellular domain. The CRLR-RAMP2 complex exhibits several intermolecular interactions that were not observed in the previously reported CRLR-RAMP1 complex, and thus the shape of the putative ligand-binding pocket of CRLR-RAMP2 is distinct from that of CRLR-RAMP1. The CRLR-RAMP2 interactions were confirmed for the full-length proteins on the cell surface by site-specific photo-crosslinking. Mutagenesis revealed that AM binding requires RAMP2 residues that are not conserved in RAMP1. Therefore, the differences in both the shapes and the key residues of the binding pocket are essential for the ligand specificity.
Protein Science 11/2011; 21(2):199-210. · 2.80 Impact Factor
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Mutsuko Kukimoto-Niino,
Alexander Tokmakov,
Takaho Terada,
Naomi Ohbayashi,
Takako Fujimoto,
Sumiko Gomi,
Ikuya Shiromizu,
Masaki Kawamoto,
Tomokazu Matsusue,
Mikako Shirouzu,
Shigeyuki Yokoyama
[show abstract]
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ABSTRACT: The mitochondrial pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. PDC activity is tightly regulated by four members of a family of pyruvate dehydrogenase kinase isoforms (PDK1-4), which phosphorylate and inactivate PDC. Recently, the development of specific inhibitors of PDK4 has become an especially important focus for the pharmaceutical management of diabetes and obesity. In this study, crystal structures of human PDK4 complexed with either AMPPNP, ADP or the inhibitor M77976 were determined. ADP-bound PDK4 has a slightly wider active-site cleft and a more disordered ATP lid compared with AMPPNP-bound PDK4, although both forms of PDK4 assume open conformations with a wider active-site cleft than that in the closed conformation of the previously reported ADP-bound PDK2 structure. M77976 binds to the ATP-binding pocket of PDK4 and causes local conformational changes with complete disordering of the ATP lid. M77976 binding also leads to a large domain rearrangement that further expands the active-site cleft of PDK4 compared with the ADP- and AMPPNP-bound forms. Biochemical analyses revealed that M77976 inhibits PDK4 with increased potency compared with the previously characterized PDK inhibitor radicicol. Thus, the present structures demonstrate for the first time the flexible and dynamic aspects of PDK4 in the open conformation and provide a basis for the development of novel inhibitors targeting the nucleotide-binding pocket of PDK4.
Acta crystallographica. Section D, Biological crystallography 09/2011; 67(Pt 9):763-73. · 12.67 Impact Factor
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Mutsuko Kukimoto-Niino,
Seiko Yoshikawa,
Tetsuo Takagi,
Noboru Ohsawa,
Yuri Tomabechi,
Takaho Terada,
Mikako Shirouzu,
Atsushi Suzuki,
Suni Lee,
Toshimasa Yamauchi,
Miki Okada-Iwabu,
Masato Iwabu,
Takashi Kadowaki,
Yasuhiko Minokoshi,
Shigeyuki Yokoyama
[show abstract]
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ABSTRACT: Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) kinase (CaMKK) is a member of the CaMK cascade that mediates the response to intracellular Ca(2+) elevation. CaMKK phosphorylates and activates CaMKI and CaMKIV, which directly activate transcription factors. In this study, we determined the 2.4 Å crystal structure of the catalytic kinase domain of the human CaMKKβ isoform complexed with its selective inhibitor, STO-609. The structure revealed that CaMKKβ lacks the αD helix and that the equivalent region displays a hydrophobic molecular surface, which may reflect its unique substrate recognition and autoinhibition. Although CaMKKβ lacks the activation loop phosphorylation site, the activation loop is folded in an active-state conformation, which is stabilized by a number of interactions between amino acid residues conserved among the CaMKK isoforms. An in vitro analysis of the kinase activity confirmed the intrinsic activity of the CaMKKβ kinase domain. Structure and sequence analyses of the STO-609-binding site revealed amino acid replacements that may affect the inhibitor binding. Indeed, mutagenesis demonstrated that the CaMKKβ residue Pro(274), which replaces the conserved acidic residue of other protein kinases, is an important determinant for the selective inhibition by STO-609. Therefore, the present structure provides a molecular basis for clarifying the known biochemical properties of CaMKKβ and for designing novel inhibitors targeting CaMKKβ and the related protein kinases.
Journal of Biological Chemistry 06/2011; 286(25):22570-9. · 4.77 Impact Factor
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ABSTRACT: RH-RhoGEFs are a family of guanine nucleotide exchange factors that contain a regulator of G protein signaling homology (RH) domain. The heterotrimeric G protein Gα(13) stimulates the guanine nucleotide exchange factor (GEF) activity of RH-RhoGEFs, leading to activation of RhoA. The mechanism by which Gα(13) stimulates the GEF activity of RH-RhoGEFs, such as p115RhoGEF, has not yet been fully elucidated. Here, specific residues in Gα(13) that mediate activation of p115RhoGEF are identified. Mutation of these residues significantly impairs binding of Gα(13) to p115RhoGEF as well as stimulation of GEF activity. These data suggest that the exchange activity of p115RhoGEF is stimulated allosterically by Gα(13) and not through its interaction with a secondary binding site. A crystal structure of Gα(13) bound to the RH domain of p115RhoGEF is also presented, which differs from a previously crystallized complex with a Gα(13)-Gα(i1) chimera. Taken together, these data provide new insight into the mechanism by which p115RhoGEF is activated by Gα(13).
Journal of Biological Chemistry 06/2011; 286(23):20625-36. · 4.77 Impact Factor
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[show abstract]
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ABSTRACT: RH-RhoGEFs are a family of guanine nucleotide exchange factors that contain a regulator of G protein signaling homology (RH)
domain. The heterotrimeric G protein Gα13 stimulates the guanine nucleotide exchange factor (GEF) activity of RH-RhoGEFs, leading to activation of RhoA. The mechanism
by which Gα13 stimulates the GEF activity of RH-RhoGEFs, such as p115RhoGEF, has not yet been fully elucidated. Here, specific residues
in Gα13 that mediate activation of p115RhoGEF are identified. Mutation of these residues significantly impairs binding of Gα13 to p115RhoGEF as well as stimulation of GEF activity. These data suggest that the exchange activity of p115RhoGEF is stimulated
allosterically by Gα13 and not through its interaction with a secondary binding site. A crystal structure of Gα13 bound to the RH domain of p115RhoGEF is also presented, which differs from a previously crystallized complex with a Gα13-Gαi1 chimera. Taken together, these data provide new insight into the mechanism by which p115RhoGEF is activated by Gα13.
Journal of Biological Chemistry 06/2011; 286(23):20625-20636. · 4.77 Impact Factor
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Daisuke Morishita,
Miho Takami,
Seiko Yoshikawa,
Ryohei Katayama,
Shigeo Sato, Mutsuko Kukimoto-Niino,
Takashi Umehara,
Mikako Shirouzu,
Kazuhisa Sekimizu,
Shigeyuki Yokoyama,
Naoya Fujita
[show abstract]
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ABSTRACT: The incidence and death rate of prostate cancer is increasing rapidly. In addition, the low sensitivity of prostate cancer to chemotherapy makes it difficult to treat this condition. The serine/threonine kinase Pim-1 plays an important role in cell cycle progression and apoptosis inhibition, resulting in prostate tumorigenesis. Therefore, Pim-1 inhibition has been expected to be an attractive target for developing new anti-cancer drugs. However, no small compounds targeting Pim-1 have progressed to clinical use because of their lack of specificity. Here, we have reported a new cell-permeable Pim-1 inhibitory p27(Kip1) peptide that could interfere with the binding of Pim-1 to its substrates and act as an anti-cancer drug. The peptide could bind to Pim-1 and inhibit phosphorylation of endogenous p27(Kip1) and Bad by Pim-1. Treatment of prostate cancer with the peptide induces G(1) arrest and subsequently apoptosis in vitro. However, the peptide showed almost no growth inhibitory or apoptosis-inducing effects in normal cells. The peptide could inhibit tumor growth in in vivo prostate cancer xenograft models. Moreover, the peptide treatment could overcome resistance to taxol, one of the first line chemotherapeutic agents for prostate cancer, and a combination of the peptide with taxol synergistically inhibited prostate cancer growth in vivo. These results indicate that a Pim-1 inhibitory p27(Kip1) peptide could be developed as an anti-cancer drug against prostate cancer.
Journal of Biological Chemistry 11/2010; 286(4):2681-8. · 4.77 Impact Factor
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ABSTRACT: The 70 kDa heat-shock proteins (Hsp70s) are highly conserved chaperones that are involved in several cellular processes, such as protein folding, disaggregation and translocation. In this study, the crystal structures of the human Hsp70 nucleotide-binding domain (NBD) fragment were determined in the nucleotide-free state and in complex with adenosine 5'-(beta,gamma-imido)triphosphate (AMPPNP). The structure of the nucleotide-free NBD fragment is similar to that of the AMPPNP-bound NBD fragment and is designated as the ;closed form'. In the nucleotide-free NBD fragment the closed form is intrinsically supported through interactions between Tyr15, Lys56 and Glu268 which connect subdomains IA, IB and IIB at the centre of the protein. Interaction with the substrate-binding domain (SBD) of Hsp70 or the BAG domain of BAG1 impairs this subdomain connection and triggers the rotation of subdomain IIA around a hydrophobic helix from subdomain IA. The subdomain rotation is limited by Asp199 and Asp206 from subdomain IIA and clearly defines the open form of the NBD. The open form is further stabilized by a new interaction between Gly230 from subdomain IIB and Ser340 from subdomain IIA. The structure of the NBD in the nucleotide-free state is determined by switching of the inter-subdomain interactions.
Acta crystallographica. Section D, Biological crystallography 03/2010; 66(Pt 3):223-32. · 12.67 Impact Factor
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Toshiyuki Kawashima,
Ying Chun Bao,
Yukinori Minoshima,
Yasushi Nomura,
Tomonori Hatori,
Tetsuya Hori,
Tatsuo Fukagawa,
Toshiyuki Fukada,
Noriko Takahashi,
Tetsuya Nosaka,
Makoto Inoue,
Tomohiro Sato, Mutsuko Kukimoto-Niino,
Mikako Shirouzu,
Shigeyuki Yokoyama,
Toshio Kitamura
[show abstract]
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ABSTRACT: In addition to their pleiotropic functions under physiological conditions, transcription factors STAT3 and STAT5 also have oncogenic activities, but how activated STATs are transported to the nucleus has not been fully understood. Here we show that an MgcRacGAP mutant lacking its nuclear localizing signal (NLS) blocks nuclear translocation of p-STATs both in vitro and in vivo. Unlike wild-type MgcRacGAP, this mutant did not promote complex formation of phosphorylated STATs (p-STATs) with importin alpha in the presence of GTP-bound Rac1, suggesting that MgcRacGAP functions as an NLS-containing nuclear chaperone. We also demonstrate that mutants of STATs lacking the MgcRacGAP binding site (the strand betab) are hardly tyrosine phosphorylated after cytokine stimulation. Intriguingly, mutants harboring small deletions in the C'-adjacent region (betab-betac loop region) of the strand betab became constitutively active with the enhanced binding to MgcRacGAP. The molecular basis of this phenomenon will be discussed, based on the computer-assisted tertiary structure models of STAT3. Thus, MgcRacGAP functions as both a critical mediator of STAT's tyrosine phosphorylation and an NLS-containing nuclear chaperone of p-STATs.
Molecular and cellular biology 02/2009; 29(7):1796-813. · 6.06 Impact Factor
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ABSTRACT: Rab27A is required for actin-based melanosome transport in mammalian skin melanocytes through its interaction with a specific effector, Slac2-a/melanophilin. Mutations that disrupt the Rab27A/Slac2-a interaction cause human Griscelli syndrome. The other Rab27 isoform, Rab27B, also binds all of the known effectors of Rab27A. In this study, we determined the crystal structure of the constitutively active form of Rab27B complexed with GTP and the effector domain of Slac2-a. The Rab27B/Slac2-a complex exhibits several intermolecular hydrogen bonds that were not observed in the previously reported Rab3A/rabphilin complex. A Rab27A mutation that disrupts one of the specific hydrogen bonds with Slac2-a resulted in the dramatic reduction of Slac2-a binding activity. Furthermore, we generated a Rab3A mutant that acquires Slac2-a binding ability by transplanting four Rab27-specific residues into Rab3A. These findings provide the structural basis for the exclusive association of Slac2-a with the Rab27 subfamily, whereas rabphilin binds several subfamilies, including Rab3 and Rab27.
Structure 11/2008; 16(10):1478-90. · 6.35 Impact Factor
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ABSTRACT: Receptor activity-modifying protein (RAMP) 1 forms a heterodimer with calcitonin receptor-like receptor (CRLR) and regulates its transport to the cell surface. The CRLR.RAMP1 heterodimer functions as a specific receptor for calcitonin gene-related peptide (CGRP). Here, we report the crystal structure of the human RAMP1 extracellular domain. The RAMP1 structure is a three-helix bundle that is stabilized by three disulfide bonds. The RAMP1 residues important for cell-surface expression of the CRLR.RAMP1 heterodimer are clustered to form a hydrophobic patch on the molecular surface. The hydrophobic patch is located near the tryptophan residue essential for binding of the CGRP antagonist, BIBN4096BS. These results suggest that the hydrophobic patch participates in the interaction with CRLR and the formation of the ligand-binding pocket when it forms the CRLR.RAMP1 heterodimer.
Protein Science 09/2008; 17(11):1907-14. · 2.80 Impact Factor
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Shaun K Olsen,
Naruhisa Ota,
Seiichiro Kishishita, Mutsuko Kukimoto-Niino,
Kazutaka Murayama,
Hidemi Uchiyama,
Mitsutoshi Toyama,
Takaho Terada,
Mikako Shirouzu,
Osami Kanagawa,
Shigeyuki Yokoyama
[show abstract]
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ABSTRACT: Interleukin (IL)-15 is a pleiotropic cytokine that plays a pivotal role in both innate and adaptive immunity. IL-15 is unique among cytokines due to its participation in a trans signaling mechanism in which IL-15 receptor alpha (IL-15Ralpha) from one subset of cells presents IL-15 to neighboring IL-2Rbeta/gammac-expressing cells. Here we present the crystal structure of IL-15 in complex with the sushi domain of IL-15Ralpha. The structure reveals that the alpha receptor-binding epitope of IL-15 adopts a unique conformation, which, together with amino acid substitutions, permits specific interactions with IL-15Ralpha that account for the exceptionally high affinity of the IL-15.IL-15Ralpha complex. Interestingly, analysis of the topology of IL-15 and IL-15Ralpha at the IL-15.IL-15Ralpha interface suggests that IL-15 should be capable of participating in a cis signaling mechanism similar to that of the related cytokine IL-2. Indeed, we present biochemical data demonstrating that IL-15 is capable of efficiently signaling in cis through IL-15Ralpha and IL-2Rbeta/gammac expressed on the surface of a single cell. Based on our data we propose that cis presentation of IL-15 may be important in certain biological contexts and that flexibility of IL-15Ralpha permits IL-15 and its three receptor components to be assembled identically at the ligand-receptor interface whether IL-15 is presented in cis or trans. Finally, we have gained insights into IL-15.IL-15Ralpha.IL-2Rbeta.gammac quaternary complex assembly through the use of molecular modeling.
Journal of Biological Chemistry 01/2008; 282(51):37191-204. · 4.77 Impact Factor
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Shaun K. Olsen,
Naruhisa Ota,
Seiichiro Kishishita, Mutsuko Kukimoto-Niino,
Kazutaka Murayama,
Hidemi Uchiyama,
Mitsutoshi Toyama,
Takaho Terada,
Mikako Shirouzu,
Osami Kanagawa,
Shigeyuki Yokoyama
[show abstract]
[hide abstract]
ABSTRACT: Interleukin (IL)-15 is a pleiotropic cytokine that plays a pivotal role in both innate and adaptive immunity. IL-15 is unique
among cytokines due to its participation in a trans signaling mechanism in which IL-15 receptorα (IL-15Rα) from one subset of cells presents IL-15 to neighboring IL-2Rβ/γc-expressing cells. Here we present the crystal structure of IL-15 in complex with the sushi domain of IL-15Rα. The structure
reveals that theα receptor-binding epitope of IL-15 adopts a unique conformation, which, together with amino acid substitutions,
permits specific interactions with IL-15Rα that account for the exceptionally high affinity of the IL-15·IL-15Rα complex.
Interestingly, analysis of the topology of IL-15 and IL-15Rα at the IL-15·IL-15Rα interface suggests that IL-15 should be
capable of participating in a cis signaling mechanism similar to that of the related cytokine IL-2. Indeed, we present biochemical data demonstrating that
IL-15 is capable of efficiently signaling in cis through IL-15Rα and IL-2Rβ/γc expressed on the surface of a single cell. Based on our data we propose that cis presentation of IL-15 may be important in certain biological contexts and that flexibility of IL-15Rα permits IL-15 and its
three receptor components to be assembled identically at the ligand-receptor interface whether IL-15 is presented in cis or trans. Finally, we have gained insights into IL-15·IL-15Rα·IL-2Rβ·γc quaternary complex assembly through the use of molecular modeling.
Journal of Biological Chemistry 12/2007; 282(51):37191-37204. · 4.77 Impact Factor
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Noriko Handa, Mutsuko Kukimoto-Niino,
Ryogo Akasaka,
Kazutaka Murayama,
Takaho Terada,
Makoto Inoue,
Takashi Yabuki,
Masaaki Aoki,
Eiko Seki,
Takayoshi Matsuda,
Emi Nunokawa,
Akiko Tanaka,
Yoshihide Hayashizaki,
Takanori Kigawa,
Mikako Shirouzu,
Shigeyuki Yokoyama
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ABSTRACT: UNC5Hs (UNC5H1-4) are netrin 1 receptors that are involved in axonal guidance and neuronal migration. They are dependence receptors that mediate apoptosis in the absence of netrin 1. UNC5H2-induced apoptosis depends on the interaction of the death domain at the C-terminus with the DAP-kinase death domain and caspase cleavage near the transmembrane region. Here, the crystal structure of the mouse UNC5H2 death domain has been determined at 2.1 A resolution. The domain adopts a six-helix bundle fold, which is similar to those of the other members of the death-domain superfamily. The UNC5H2 death domain is a dimer in the crystal and in solution. This homodimerized structure may represent the structure of the death domain when netrin 1 binds to the UNC5H2 receptor. Homodimerization of UNC5H2 may block the access of caspase to the cleavage site. In the death-domain dimer, residues in alpha3 and the 3(10)-helix preceding alpha3 and the residues in alpha4 make significant contacts, mainly by hydrophobic and van der Waals interactions.
Acta Crystallographica Section D Biological Crystallography 01/2007; 62(Pt 12):1502-9. · 12.62 Impact Factor
