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Yutaka Kofuku,
Chie Yoshiura,
Takumi Ueda,
Hiroaki Terasawa,
Takahiro Hirai,
Sae Tominaga, Masako Hirose,
Yoshitake Maeda,
Hideo Takahashi,
Yuya Terashima,
Kouji Matsushima,
Ichio Shimada
[show abstract]
[hide abstract]
ABSTRACT: The chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) and its G-protein-coupled receptor (GPCR) CXCR4 play fundamental
roles in many physiological processes, and CXCR4 is a drug target for various diseases such as cancer metastasis and human
immunodeficiency virus, type 1, infection. However, almost no structural information about the SDF-1-CXCR4 interaction is
available, mainly because of the difficulties in expression, purification, and crystallization of CXCR4. In this study, an
extensive investigation of the preparation of CXCR4 and optimization of the experimental conditions enables NMR analyses of
the interaction between the full-length CXCR4 and SDF-1. We demonstrated that the binding of an extended surface on the SDF-1
β-sheet, 50-s loop, and N-loop to the CXCR4 extracellular region and that of the SDF-1 N terminus to the CXCR4 transmembrane
region, which is critical for G-protein signaling, take place independently by methyl-utilizing transferred cross-saturation
experiments along with the usage of the CXCR4-selective antagonist AMD3100. Furthermore, based upon the data, we conclude
that the highly dynamic SDF-1 N terminus in the 1st step bound state plays a crucial role in efficiently searching the deeply
buried binding pocket in the CXCR4 transmembrane region by the “fly-casting” mechanism. This is the first structural analyses
of the interaction between a full-length GPCR and its chemokine, and our methodology would be applicable to other GPCR-ligand
systems, for which the structural studies are still challenging.
Journal of Biological Chemistry 12/2009; 284(50):35240-35250. · 4.77 Impact Factor
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Yutaka Kofuku,
Chie Yoshiura,
Takumi Ueda,
Hiroaki Terasawa,
Takahiro Hirai,
Sae Tominaga, Masako Hirose,
Yoshitake Maeda,
Hideo Takahashi,
Yuya Terashima,
Kouji Matsushima,
Ichio Shimada
[show abstract]
[hide abstract]
ABSTRACT: The chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) and its G-protein-coupled receptor (GPCR) CXCR4 play fundamental roles in many physiological processes, and CXCR4 is a drug target for various diseases such as cancer metastasis and human immunodeficiency virus, type 1, infection. However, almost no structural information about the SDF-1-CXCR4 interaction is available, mainly because of the difficulties in expression, purification, and crystallization of CXCR4. In this study, an extensive investigation of the preparation of CXCR4 and optimization of the experimental conditions enables NMR analyses of the interaction between the full-length CXCR4 and SDF-1. We demonstrated that the binding of an extended surface on the SDF-1 beta-sheet, 50-s loop, and N-loop to the CXCR4 extracellular region and that of the SDF-1 N terminus to the CXCR4 transmembrane region, which is critical for G-protein signaling, take place independently by methyl-utilizing transferred cross-saturation experiments along with the usage of the CXCR4-selective antagonist AMD3100. Furthermore, based upon the data, we conclude that the highly dynamic SDF-1 N terminus in the 1st step bound state plays a crucial role in efficiently searching the deeply buried binding pocket in the CXCR4 transmembrane region by the "fly-casting" mechanism. This is the first structural analyses of the interaction between a full-length GPCR and its chemokine, and our methodology would be applicable to other GPCR-ligand systems, for which the structural studies are still challenging.
Journal of Biological Chemistry 10/2009; 284(50):35240-50. · 4.77 Impact Factor
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Michael D Feese,
Taro Tamada,
Yoichi Kato,
Yoshitake Maeda, Masako Hirose,
Yasuko Matsukura,
Hideki Shigematsu,
Takanori Muto,
Atsushi Matsumoto,
Hiroshi Watarai,
Kinya Ogami,
Tomoyuki Tahara,
Takashi Kato,
Hiroshi Miyazaki,
Ryota Kuroki
[show abstract]
[hide abstract]
ABSTRACT: The cytokine thrombopoietin (TPO), the ligand for the hematopoietic receptor c-Mpl, acts as a primary regulator of megakaryocytopoiesis and platelet production. We have determined the crystal structure of the receptor-binding domain of human TPO (hTPO(163)) to a 2.5-A resolution by complexation with a neutralizing Fab fragment. The backbone structure of hTPO(163) has an antiparallel four-helix bundle fold. The neutralizing Fab mainly recognizes the C-D crossover loop containing the species invariant residue Q111. Titration calorimetric experiments show that hTPO(163) interacts with soluble c-Mpl containing the extracellular cytokine receptor homology domains with 1:2 stoichiometry with the binding constants of 3.3 x 10(9) M(-1) and 1.1 x 10(6) M(-1). The presence of the neutralizing Fab did not inhibit binding of hTPO(163) to soluble c-Mpl fragments, but the lower-affinity binding disappeared. Together with prior genetic data, these define the structure-function relationships in TPO and the activation scheme of c-Mpl.
Proceedings of the National Academy of Sciences 03/2004; 101(7):1816-21. · 9.68 Impact Factor
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Ryota Kuroki, Masako Hirose,
Yoichi Kato,
Michael D Feese,
Taro Tamada,
Hideki Shigematsu,
Hiroshi Watarai,
Yoshitake Maeda,
Tomoyuki Tahara,
Takashi Kato,
Hiroshi Miyazaki
[show abstract]
[hide abstract]
ABSTRACT: Thrombopoietin (TPO) is a cytokine which primarily stimulates megakaryocytopoiesis and thrombopoiesis. The functional domain of TPO (TPO(163)) consisting of the N-terminal 163 amino acids was prepared and crystallized. Since the crystallization of TPO(163) was unsuccessful using the standard screening methods, a Fab fragment derived from a neutralizing monoclonal antibody was used for crystallization. It was found that the TPO(163)-Fab complex crystallized reproducibly in 0.1 M potassium phosphate buffer pH 6.0 containing 20-25% polyethylene glycol 4000. Thin crystals (0.2 x 0.2 x 0.02 mm) grew in two space groups: P2(1), with unit-cell parameters a = 133.20, b = 46.71, c = 191.47 A, beta = 90.24 degrees, and C2, with unit-cell parameters a = 131.71, b = 46.48, c = 184.63 A, beta = 90.42 degrees. The results of a molecular-replacement analysis indicate that the Fab molecules interact with each other and provide a suitable interface for crystallization.
Acta Crystallographica Section D Biological Crystallography 06/2002; 58(Pt 5):856-8. · 12.62 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: The crystal structure of glycosyltrehalose trehalohydrolase from the hyperthermophilic archaeum Sulfolobus solfataricus KM1 has been solved by multiple isomorphous replacement. The enzyme is an α-amylase (family 13) with unique exo-amylolytic activity for glycosyltrehalosides. It cleaves the α-1,4 glycosidic bond adjacent to the trehalose moiety to release trehalose and maltooligo saccharide. Unlike most other family 13 glycosidases, the enzyme does not require Ca2+ for activity, and it contains an N-terminal extension of ∼100 amino acid residues that is homologous to N-terminal domains found in many glycosidases that recognize branched oligosaccharides. Crystallography revealed the enzyme to exist as a homodimer covalently linked by an intermolecular disulfide bond at residue C298. The existence of the intermolecular disulfide bond was confirmed by biochemical analysis and mutagenesis. The N-terminal extension forms an independent domain connected to the catalytic domain by an extended linker. The functionally essential Ca2+ binding site found in the B domain of α-amylases and many other family 13 glycosidases was found to be replaced by hydrophobic packing interactions. The enzyme also contains a very unusual excursion in the (β/α)8 barrel structure of the catalytic domain. This excursion originates from the bottom of the (β/α)8 barrel between helix 6 and strand 7, but folds upward in a distorted α-hairpin structure to form a part of the substrate binding cleft wall that is possibly critical for the enzyme’s unique substrate selectivity. Participation of an α-β loop in the formation of the substrate binding cleft is a novel feature that is not observed in other known (β/α)8 enzymes.
Journal of Molecular Biology.
