3D分子設計チーム

重点課題1  重点課題2

研究詳細 : 重点課題2

担当:竹内 恒 研究員

核磁気共鳴(NMR)法は生理的条件下における生体高分子の立体構造情報を提供する分光法です。

解析対象タンパク質を含む水溶液試料に任意の結合リガンドを添加し、そのNMRスペクトル変化から定量性のある、原子レベルの相互作用情報を得ることが 可能な測定法であるといえます。
その一方で、高感度・高精度の解析を行うため安定同位体を試料に取りこませ、かつ凝集のない状態で測定しなければならないことや、 解析対象分子量が巨大になると測定が困難になる、といった制約があります。

我々は、NMRによる膜タンパク質等高分子量タンパク質-リガンド複合体の相互作用解析のため、適切な試料調製方法の確立や、測定技術開発、 および創薬標的タンパク質を対象とした実証研究を行うとともに、NMR相互作用解析情報を活用した機能性分子創出技術の開発も進めています。

概念図

高分子量タンパク質複合体の相互作用部位を
高精度に同定する転移交差飽和法の概念図

 

KcsAカリウムチャネルと阻害分子のドッキングモデル

転移交差飽和法のデータを利用し計算した
KcsAカリウムチャネルと阻害分子のドッキングモデル

 

最近の主な成果

NMRと計算の複合によるタンパク質‐化合物複合体構造予測技術

NMRによる疾患関連タンパク質-リガンド相互作用解析

 

研究成果、論文リスト等

2016
Use of multiple cryoprotectants to improve diffraction quality from protein crystals.
Cryst Growth Des, 16, 1565–1571, (2016).
Senda M, Hayashi T, Hatakeyama M, Takeuchi K, Sasaki AT, Senda T.

Structural reverse genetics study of the PI5P4Kβ-nucleotide complexes reveals the presence of the GTP bioenergetic system in mammalian cells.
FEBS J, 283, 3556–3562, (2016).
Takeuchi K, Senda M, Lo YH, Kofuji S, Ikeda Y, Sasaki AT, Senda T.

Improvement of affinity and thermodynamic properties of weak-affinity ligands by NMR-based evaluation of local dynamics and surface complementarity in the bound state.
Angew Chem Int Ed, 55, 14606-14609, (2016).
*Mizukoshi Y, *Takeuchi K, Arutaki M, Tokunaga Y, Takizawa T, Hanzawa H, Shimada I, (*equal contribution).

Revisiting the Field Dependence of TROSY Sensitivity
J Biomol NMR, 66, 221-225, (2016).
Takeuchi K, Arthanari H, Wagner G

The Lipid Kinase PI5P4Kβ Is an Intracellular GTP Sensor for Metabolism and Tumorigenesis.
Molecular Cell, 61, 187–198, (2016).
*Sumita K, *Lo TH, *Takeuchi K, Senda M, Kofuji S, Ikeda Y, Terakawa J, Sasaki M, Yoshino H, Majd N, Zheng Y, Kahoud ER, Yokota T, Emerling BM, Asara JM, Ishida T, Locasale JW, Daikoku T, Anastasiou D, Senda T, Sasaki AT. (*equal contribution).

Nitrogen-detected TROSY yields comparable sensitivity to proton-detected TROSY for non-deuterated, large proteins under physiological salt conditions.
J Biomol NMR,  64, 143-151, (2016).
Takeuchi K, Arthanari H, Imai M, Wagner G, Shimada I.

2015
Suppression of problematic compound oligomerization by cosolubilization of nondetergent sulfobetaines.
ChemMedChem, 10, 736-741, (2015).
Mizukoshi Y, Takeuchi K, Arutaki M, Takizawa T, Hanzawa H, Takahashi H, Shimada I

Development of a method for reconstruction of crowded NMR spectra from undersampled time-domain data.
J Biomol NMR, 62, 31-41, (2015).
Ueda T, Yoshiura C, Matsumoto M, Kofuku Y, Okude J, Kondo K, Shiraishi Y, Takeuchi K, Shimada I.

Increased resolution of aromatic cross peaks using alternate 13C labeling and TROSY Milbradt AG
J Biomol NMR, 62, 291-301, (2015).
Arthanari H, Takeuchi K, Boeszoermenyi A, Hagn F, Wagner G.

Nitrogen detected TROSY at high field yields high resolution and sensitivity for protein NMR.
J Biomol NMR, 63, 323-331, (2015).
Takeuchi K, Arthanari H, *Shimada I, *Wagner G.

2014
Structure-based approach to improve a small-molecule inhibitor by the use of a competitive Peptide ligand.
Angew Chem Int Ed Engl.2014 Mar 3 ;53(10):2597-601.doi: 10.1002/anie.201310749.
Epub 2014 Jan 30.
Ono K, Takeuchi K, Ueda H, Morita Y, Tanimura R, Shimada I, Takahashi H

2013
Degradation of Activated K-Ras Orthologue via K-Ras Specific Lysine Residues is Required for Cytokinesis.
J Biol Chem. 2013 289(7):3950-9.
Sumita K, Yoshino H, Sasaki M, Majd N, Kahoud ER, Takahashi H, Takeuchi K, Kuroda T, Lee S, Charest PG, Takeda K, Asara JM, Firtel RA, Anastasiou D, Sasaki AT.

Functional dynamics of cell surface membrane proteins
J Magn Reson. 2013 Nov 22. pii: S1090-7807(13)00290-5. doi: 10.1016/j.jmr.2013.11.007.
Nishida N, Osawa M, Takeuchi K, Imai S, Stampoulis P, Kofuku Y, Ueda T, Shimada I

Rapid identification of ligand-binding sites by using an assignment-free NMR approach.
J Med Chem. 2013 Nov 27;56(22):9342-50. doi: 10.1021/jm4014357. Epub 2013 Nov 13.
Kodama Y, Takeuchi K, Shimba N, Ishikawa K, Suzuki E, Shimada I, Takahashi H.

Perdeuteration and methyl-selective 1H, 13C-labeling by using a Kluyveromyces lactis expression system.
J Biomol NMR, 2013, 57, 297-304
Miyazawa-Ohnami M, Takeuchi K, Takano T, Sugiki T, Shimada I, Takahashi H

溶液NMR法による膜タンパク質の機能調節メカニズムの解明
生物物理. 有、2013, 53, 236-241
大澤匡範,今井駿輔,竹内 恒,嶋田一夫

2012
An accurate pharmacophore mapping method by NMR spectroscopy.
Angew Chem Int Ed Engl. 51, 1362-5.
Mizukoshi, Y., Abe, A., Takizawa, T., Hanzawa, H., Fukunishi, Y., Shimada, I. and Takahashi, H.

Backbone resonance assignments for the cytoplasmic region of the Mg2+ transporter MgtE in the Mg2+-unbound state.
Biomol NMR Assign. in press.
Maruyama, T., Imai, S., Osawa, M., Hattori, M., Ishitani, R., Nureki, O. and Shimada, I.

Structural basis for the modulation of the gating property of G protein-gated inwardly rectifying potassium ion channel (GIRK) by the i/o-family G protein α subunit (Gα i/o ).
J Biol Chem. in press.
Mase, Y., Yokogawa, M., Osawa, M. and Shimada, I.

Backbone resonance assignments for G protein α (i3)subunit in the GTP-bound state.
Biomol NMR Assign. in press.
Mase, Y., Yokogawa, M., Osawa, M. and Shimada, I.

Atypical membrane-embedded PI(3,4)P2 binding site on p47phox PX domain revealed by NMR.
J Biol Chem. in press.
Stampoulis, P., Ueda, T., Matsumoto, M., Terasawa, H., Miyano, K., Sumimoto, H. and Shimada, I.

2011
Protein-ligand docking guided by ligand pharmacophore-mapping experiment by NMR.
J Mol Graph Model. 31, 20-7.
Fukunishi, Y., Mizukoshi, Y., Takeuchi, K., Shimada, I., Takahashi, H. and Nakamura, H.

Speeding up direct 15N detection: hCaN 2D NMR experiment.
J Biomol NMR. 51, 497-504.
Gal, M., Edmonds, K. A., Milbradt, A. G., Takeuchi, K. and Wagner, G.

Affinity transfer to a human protein by CDR3 grafting of camelid VHH.
Protein Sci. 20, 1971-81.
Inoue, H., Iihara, A., Takahashi, H., Shimada, I., Ishida, I. and Maeda, Y.

Rapid identification of protein-protein interfaces for the construction of a complex model based on multiple unassigned signals by using time-sharing NMR measurements.
J Struct Biol. 174, 434-42.
Kodama Y, Reese ML, Shimba N, Ono K, Kanamori E, Dötsch V, Noguchi S, Fukunishi Y, Suzuki EI, Shimada I, Takahashi H.

Ubiquitination of K-Ras enhances activation and facilitates binding to select downstream effectors.
Sci Signal. 4, ra13.
Sasaki AT, Carracedo A, Locasale JW, Anastasiou D, Takeuchi K, Kahoud ER, Haviv S, Asara JM, Pandolfi PP, Cantley LC.

Isotopic labeling of heterologous proteins in the yeast Pichia pastoris and Kluyveromyces lactis.
Methods Mol Biol. 831, 19-36.
Sugiki, T., Ichikawa, O., Miyazawa-Onami, M., Shimada, I. and Takahashi, H.

2010
HNCA-TOCSY-CANH experiments with alternate (13)C- (12)C labeling: a set of 3D experiment with unique supra-sequential information for mainchain resonance assignment.
J Biomol NMR. 49, 17-26.
Takeuchi K, Gal M, Takahashi H, Shimada I, Wagner G.

NMR analyses of the Gbg binding and conformational rearrangements of the cytoplasmic pore of G protein-activated inwardly rectifying potassium channel 1 (GIRK1).
J Biol Chem. 286, 2215-23.
Yokogawa, M., Osawa, M., Takeuchi, K., Mase, Y. and Shimada, I.

Precise structural determination of weakly binding peptides by utilizing dihedral angle constraints.
J Biomol NMR. 46, 299-305.
Mizukoshi Y, Nagasu M, Shimada I, Takahashi H.

Structural basis for platelet antiaggregation by angiotensin II type 1 receptor antagonist losartan (DuP-753) via glycoprotein VI.
J Med Chem. 53, 2087-93.
Ono K, Ueda H, Yoshizawa Y, Akazawa D, Tanimura R, Shimada I, Takahashi H.

Direct determination of the insulin-insulin receptor interface using transferred cross-saturation experiments.
J Med Chem. 53, 1917-22.
Nakamura T, Takahashi H, Takahashi M, Shimba N, Suzuki E, Shimada I.

Real-time assay method of lipid extraction activity.
Anal Biochem. 399, 162-7.
Sugiki T, Takahashi H, Nagasu M, Hanada K, Shimada I.

Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells.
J Biomol NMR. 46, 3-10.
Takahashi H, Shimada I.

2009
Structural basis of the interaction between chemokine stromal cell-derived factor-1/CXCL12 and its G-protein-coupled receptor CXCR4.
J Biol Chem. 284, 35240-50.
Kofuku Y, Yoshiura C, Ueda T, Terasawa H, Hirai T, Tominaga S, Hirose M, Maeda Y, Takahashi H, Terashima Y, Matsushima K, Shimada I.

Structural investigation of the interaction between LolA and LolB using NMR.
J Biol Chem. 284, 24634-43.
Nakada S, Sakakura M, Takahashi H, Okuda S, Tokuda H, Shimada I.

High-throughput screening of optimal solution conditions for structural biological studies by fluorescence correlation spectroscopy.
Protein Sci. 18, 1115-20.
Sugiki T, Yoshiura C, Kofuku Y, Ueda T, Shimada I, Takahashi H.

Structural and interaction analysis of glycoprotein VI-binding peptide selected from a phage display library.
J Biol Chem. 284, 10720-7.
Kato-Takagaki K, Mizukoshi Y, Yoshizawa Y, Akazawa D, Torii Y, Ono K, Tanimura R, Shimada I, Takahashi H.

2008
Stable isotope labeling of protein by Kluyveromyces lactis for NMR study.
J. Biol. NMR. 42, 159-62.
Sugiki T, Shimada I, Takahashi H.

Cross-saturation and transferred cross-saturation experiments.
Prog. NMR Spectrosc. 54, 123-140.
Shimada I, Ueda T, Matsumoto M, Sakakura M, Osaswa M, Takeuchi K, Nishida N, and Takahashi H.

2007
Identification and characterization of the slowly exchanging pH-dependenct conformational rearrangement in KcsA.
J. Biol. Chem. 282, 15179-15186.
K. Takeuchi, H. Takahashi, S. Kawano and I. Shimada

Structural basis of the transition-state stabilization in antibody-catalyzed hydrolysis.
J. Mol. Biol. 367, 133-147.
M. Sakakura, H. Takahashi, N. Shimba, I. Fujii and I. Shimada

Pairwise NMR experiments for the determination of protein backbone dihedral angle Φ based on cross-correlated spin relaxation.
J. Biomol. NMR. 37, 179-185.
H. Takahashi and I. Shimada

Backbone resonance assignments for the periplasmic chaperone LolA from Escherichia coli.
Biomol. NMR Assign. 1, 125-127.
S. Nakada, H. Takahashi, M. Sakakura, M. Kurono and I. Shimada

Backbone resonance assignment for the outer membrane lipoprotein receptor LolB from Escherichia coli.
Biomol. NMR Assign. 1, 121-123.
S. Nakada, M. Sakakura, H. Takahashi, H. Tokuda and I. Shimada

2006
Utilization of methyl proton resonances in cross-saturation measurement for determining the interface of large protein-protein complexes.
J. Biomol. NMR. 34, 167-177.
H. Takahashi, M. Miyazawa, Y. Ina, Y. Fukunishi, Y. Mizukoshi, H. Nakamura and I. Shimada

Rapid preparation of stable isotope labeled peptides that bind to target proteins by phage library system.
J. Biomol. NMR. 34, 23-30.
Y. Mizukoshi, H. Takahashi and I. Shimada

2005
Direct determination of a membrane-peptide interface using the nuclear magnetic resonance cross-saturation method.
Biophys. J. 89 4051-4055.
T. Nakamura, H. Takahashi, K. Takeuchi, T. Kohno, K. Wakamatsu and I. Shimada

Backbone Resonance Assignment for the Fv Fragment of Catalytic Antibody 6D9 Complexed with a Transition State Analogue.
J. Biomol. NMR. 33, 282.
M. Sakakura, H. Takahashi, H. Terasawa, K. Takeuchi, I. Fujii and I. Shimada

Conformational dynamics of complementary determining region H3 of an anti-dansyl Fv fragment in the presence of its hapten.
J. Mol. Biol. 351 627-640.
M. Nakasako, T. Oka, M. Masumo, H. Takahashi, I. Shimada, Y. Yamaguchi, K. Kato and Y. Arata

2004
Channel-forming membrane permeabilization by an antibacterial protein, sapecin: determination of membrane-buried and oligomerization surfaces by NMR.
Biochem. J. 279, 4981-4987.
K. Takeuchi, H. Takahashi, M. Sugai, H. Iwai, T. Kohno, K. Sekimizu, S. Natori and I. Shimada

Molecular basis of the high-affinity activation of type 1 ryanodine receptors by imperatoxin A.
J. Biol. Chem. 377, 385-394.
C. W. Lee, E. H. Lee, K. Takeuchi, H. Takahashi, I. Shimada, K. Sato, S. Y. Shin, D. H. Kim and J. I. Kim

2003
Structural basis of the KcsA K+ channel and agitoxin2 pore-blocking toxin interaction by using the transferred cross-saturation method.
Structure. 11, 1381-1392.
K. Takeuchi, M. Yokogawa, T. Matsuda, M. Sugai, S. Kawano, T. Kohno, H. Nakamura, H. Takahashi and I. Shimada

Novel collagen-binding mode of the VWA domain determined by a transferred cross-saturation experiment.
Nat. Struct. Biol. 10, 53-58.
N. Nishida, H. Sumikawa, M. Sakakura, N. Shimba, H. Takahashi, H. Terasawa, E. Suzuki and I. Shimada

2002
Backbone 1H, 13C, 15N resonance assignments of the von Willebrand factor A3 domain.
J. Biomol. NMR. 24, 357-358.
N. Nishida, M. Miyazawa, H. Sumikawa, M. Sakakura, N. Shimba, H. Takahashi, H. Terasawa, E. Suzuki and I. Shimada

Solution structure of ω-grammotoxin SIA, a gating modifier of P/Q and N-type Ca2+ channel.
J. Mol. Biol. 321, 517-526.
K. Takeuchi, E. J. Park, C. W. Lee, J. I. Kim, H. Takahashi, K. J. Swartz and I. Shimada

Determination of the interface of a large protein complex by transferred cross-saturation measurements.
J. Mol. Biol. 318, 245-249.
T. Nakanishi, M. Miyazawa, M. Sakakura, H. Terasawa, H. Takahashi and I. Shimada

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