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1 pp. CD CDHA HA CD HABD NMR HABD C HA ordered O -state HA partially disordered PD -state PD-state CD HA HA CD CD A Two-state equilibrium of hyaluronan-binding domain is crucial for CD -mediated cell rolling Noritaka Nishida and Ichio Shimada Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan CD CD CD hyaluronan binding domain; HABD B CD N- B CD CD

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3 2 0 1 1年 1 0月 8 9 5 図3 図1 CD4 4の生理的役割とこれまでの HABD に関する構造生物学的知見 A リンパ球の炎症部位への移行メカニズム 1 ロー リング 2 強い接着 3 漏出 4 遊走の過程においてさまざまな細胞接着分子が関与する B CD4 4の1次構造の模式図とその リガンドであるヒアルロン酸 HA の構造 C E ヒアルロン酸結合ドメイン HABD の立体構造 C HABD リガンド非存在 下の結晶構造 D mer の HA 結合状態の NMR 構造 E 8mer の HA 結合状態の結晶構造 図2 HABD の2状態平衡の証明 A ヒアルロン酸非存在下および C 存在下の HSQC スペクトルで観測されるメジャーピーク とマイナーピークの強度比の温度依存性 G1 4 1と T5 9についてメジャーピークに対するマイナーピークの割合を示す B HA 非存 在下において取得した ZZ-exchange スペクトル 黒 混合時間0ms 赤 混合時間5 0 0ms D 明らかとなったリガンド存在下 非存在下における HABD の2状態平衡 矢印の大きさは各状態の平衡の偏りを示す 図3 PD-state を安定化した変異体の作製 A 単独状態の HABD の結晶構造で観測される リガンド結合部位 R4 1 から C 末 端領域にかけての相互作用ネットワーク B Y1 1A 変異体 赤 と野生型 HABD 黒 の1H-1 5N HSQC スペクトルの重ね合わせ C Y1 1A 変異体と野生型 HABD リガンド非存在下 O-state および D Y1 1A 変異体と野生型 HABD リガンド存在下 PD-state の HSQC スペクトルの間の化学シフト差の算出 E 野生型および F Y1 1A 変異体の SPR 法による HA 親和性の算出 F 野生型 と Y1 1A 変異体の SPR センサーグラムにおける解離相の比較

4 CD HA CD HABD CD HABD N C B CD LYVE- HABD NMR CD HABD NMR X C CD HABD α β B B N C HABD HA CD HABD HA HABD D HABD HA Gly C C NOE C C C ordered O state HA C partially disordered PD state O-state PD-state mer HA HABD HA HABD HA NMR HA HABD HA HABD NMR E O-state HA NMR C HA HA HA E HABD NMR HABD NMR HABD HSQC HABD NMR A NMR ZZ-exchange HABD B HABD ZZ-exchange ms HA HABD HSQC C HABD HABD O-state PD-state HA HA O-state C HABD HA O-state PD-state HA O-state HA PD-state D HABD HA HA NMR NMR HA HABD PD-state O-state NMR

5 PD-state X C PD-state C O-state O-state HA HABD O-state PD-state NMR HA CD HABD HA O-state PD-state HABD CD CD O-state PD-state O-state α C PD-state A C Y α E L O-state Y Y A HABD Y A H- NHSQC HA HABD HABD HA G T Y A B Y A HSQC HABD HSQC C HABD HSQC D Y A HA HABD PD-state NOE Y A C PD-state Y A HABD HABD HA SPR E F Kd µm Y A µm Y A HABD HABD HA O- state PD-state Y A PD O-state PD-state HA SPR Y A PD-state O-state HA koff G PD-state O-state HABD CD HABD CD CD VMRC-LCD Y A CD CD A CD Y A FITC HA FCM HA HABD HA B CD HA CD VMRC-LCD Y A C Y A CD HA multivalent HA CD CD shear stress Shear stress

6 Y A A Y A VMRC-LCD FCM Hermes VMRC-LCD B FITC HA FCM C HA Y A D HA Y A shear stress µm E Detachment assay F G Transient tether F G Cellular koff H dyn cm dyn cm

7 CD HA Y A HA µm µm shear stress Y A PD-state Y A D shear stress Y A dyn cm O-state PD-state Y A HA CD Y A E dyn cm CD µm s µm s Y A µm s shear stress HA Y A Transient tether Y A HABD HA transient tether multivalent transient tether tether transient tether kon tether transient tether HA koff mer HA CD dyn cm tether tether tether Y A tether PD-state O-state PD-state F tether cellular koff s Y A s Y A koff G Y A koff SPR PD-state HA transient tether HABD tether PD-state Y A O-state HABD HA CD HA HABD cellular koff PD-state NMR CD O-state PD-state PD-state O-state HA H NMR O-state PD-state ms transient tether CD -HA τ koff HABD HA-CD HA O-state HA-CD PD-state Y A Y A HA O-state PD-state CD O-state PD-state

8 FimH P-selectin CD EGF A PSGL- P-selectin P-selectin PSGL- CD Lectin EGF-like B shear stress C CD FN FT A CD CD catch bond shear stress shear stress dyn cm µm pn slip bond catch bond Catch bond shear stress Catch bond AFM Catch bond B EGF CD PD-state C CD CD HA PD-state catch bond

9 C CD HABD O-state PD-state NMR HA HABD NMR HA O-state PD-state HABD PD-state Y A CD X NMR CD CD R CD MT -MMP ADAM CD CD HA CD CD cancer stem cell CD HA CD Chen, S., Alon, R., Fuhlbrigge, R.C., & Springer, T.A. Proc. Natl. Acad. Sci. USA,,. Ogino, S., Nishida, N., Umemoto, R., Suzuki, M., Takeda, M., Terasawa, H., Kitayama, J., Matsumoto, M., Hayasaka, H., Miyasaka, M., & Shimada, I. Structure,,. Jackson, D.G., Prevo, R., Clasper, S., & Banerji, S. Trends Immunol.,,. Takeda, M., Terasawa, H., Sakakura, M., Yamaguchi, Y., Kajiwara, M., Kawashima, H., Miyasaka, M., & Shimada, I. J. Biol. Chem.,,. Teriete, P., Banerji, S., Noble, M., Blundell, C.D., Wright, A.J., Pickford, A.R., Lowe, E., Mahoney, D.J., Tammi, M.I., Kahmann, J.D., Campbell, I.D., Day, A.J., & Jackson, D.G. Mol. Cell,,. Takeda, M., Ogino, S., Umemoto, R., Sakakura, M., Kajiwara, M., Sugahara, K.N., Hayasaka, H., Miyasaka, M., Terasawa, H., & Shimada, I. J. Biol. Chem.,,. Banerji, S., Wright, A.J., Noble, M., Mahoney, D.J., Campbell, I.D., Day, A.J., & Jackson, D.G. Nat. Struct. Mol. Biol.,,. Sokurenko, E.V., Vogel, V., & Thomas, W.E. Cell Host. Microbe,,. Dwir, O., Kansas, G.S., & Alon, R. J. Biol. Chem.,,. Somers, W.S., Tang, J., Shaw, G.D., & Camphausen, R.T. Cell,,. Phan, U.T., Waldron, T.T., & Springer, T.A. Nat. Immunol.,,. Lou, J., Yago, T., Klopocki, A.G., Mehta, P., Chen, W., Zarnitsyna, V.I., Bovin, N.V., Zhu, C., & McEver, R.P. J. Cell. Biol.,,. Thomas, W.E., Vogel, V., & Sokurenko, E. Annu. Rev. Biophys.,,. Marshall, B.T., Long, M., Piper, J.W., Yago, T., McEver, R.P., & Zhu, C. Nature,,. Brown, R.L., Reinke, L.M., Damerow, M.S., Perez, D., Chodosh, L.A., Yang, J., & Cheng, C. J. Clin. Invest.,,. Okamoto, I., Kawano, Y., Tsuiki, H., Sasaki, J., Nakao, M., Matsumoto, M., Suga, M., Ando, M., Nakajima, M., & Saya, H. Oncogene,,. Sugahara, K.N., Murai, T., Nishinakamura, H., Kawashima, H., Saya, H., & Miyasaka, M. J. Biol. Chem.,,. Zoller, M. Nat. Rev. Cancer,,.

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