Crystal structure of human Band3 (anion exchanger)

2010 Fiscal Year Final Research Report

• Project/Area Number: 20370035
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: Kyoto University
• Principal Investigator: KOBAYASHI Takuya Kyoto University, 医学研究科, 講師 (20311730)
• Co-Investigator(Kenkyū-buntansha): MURATA Takeshi 千葉大学, 理学研究科, 特任准教授 (80415322)
• Co-Investigator(Renkei-kenkyūsha): IWATA So 京都大学, 医学研究科, 教授 (60452330)
• Project Period (FY): 2008 – 2010
• Keywords: X線結晶解析

Research Abstract

Anion exchanger (AE) gene family consists of four members, AE1-4. These AE proteins are present in most cells and regulate intracellular pH and chloride (Cl-) by the exchange transport of anions across membranes. Band 3, designated AE1, is the major integral membrane protein of the red blood cells and intercalated cells of the distal and collecting tubules of the kidney. Band 3, consisting of 911 amino acids, has two principal structural domains. Its 40 kDa N-terminal domain in the cytoplasm binds the red blood cell skeleton proteins and has a role in maintaining the shape of erythrocytes. While the membrane domain of human erythrocyte Band3 works as a Cl^-/HCO_3^- antiporter. This exchange is a key step for CO_2/O_2 circulation in the blood. In spite of their importance, structural information about Band3 is still unknown. We used X-ray crystallography to solve the three-dimensional structure of Band3, fixed in an outward-open conformation by cross-linking, at 3.4Å resolution. Band3 are formed as a dimmer in complex with Fab fragments. There are V-shaped densities near the center of the dimmer.

Research Products

• [Journal Article] Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis. (2011)
  • Author(s): H.Asada, T.Uemura, T.Yurugi-Kobayashi, M.Shiroishi, T.Shimamura, H.Tsujimoto, K.Ito, T.Sugawara, T.Nakane, N.Nomura, T.Murata, T.Haga, S.Iwata, T.Kobayashi.
  • Journal Title: Microbial Cell Factories (in press)
  • Peer Reviewed
• [Journal Article] Cloning, expression and purification of the anion exchanger 1 homologue from the basidiomycete Phanerochaete chrysosporium. (2011)
  • Author(s): N.Tokuda, K.Igarashi, T.Shimamura, T.Yurugi-Kobayashi, M.Shiroishi, K.Ito, T.Sugawara, H.Asada, T.Murata, N.Nomura, S.Iwata, T.Kobayashi.
  • Journal Title: Protein Expr. Purif. (in press)
  • Peer Reviewed
• [Journal Article] Cysteine-to-serine shuffling using a yeast expression system improves protein secretion : case of a nonglycosylated mutant of miracullin, a taste-modifying protein. (2011)
  • Author(s): K.Ito, T.Sugawara, A.Koizumi, K.Nakajima, A.Shimizu-Ibuka, M.Shiroishi, H.Asada, T.Yurugi-Kobayashi, T.Shimamura, T.Asada, T.Misaka, S.Iwata, T.Kobayashi, K.Abe.
  • Journal Title: Biotech. Lett. 33 Pages: 103-107
  • Peer Reviewed
• [Journal Article] Crystal structure of Glucansucrase form the dental caries pathogen Streptococcus mutans. (2011)
  • Author(s): K.Ito, S.Ito, T.Shimamura, S.Weyand, Y.Kawarasaki, T.Misaka, K.Abe, T.Kobayashi, A.D.Cameron, S.Iwata.
  • Journal Title: J.Mol.Biol. (in press)
  • Peer Reviewed
• [Journal Article] Crystallization and preliminary X-ray analysis of a Glucansucrase from the dental caries pathogen Streptococcus mutans. (2010)
  • Author(s): K.Ito, S.Ito, T.Shimamura, Y.Kawarasaki, T.Kobayashi, S.Iwata.
  • Journal Title: Acta. Crystallogr. Sect.F 66 Pages: 1086-1088
  • Peer Reviewed
• [Journal Article] Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin. Biochim. (2010)
  • Author(s): K.Ito, T.Sugawara, A.Koizumi, K.I.Nakajima, A.Shimizu-Ibuka, M.Shiroishi, H.Asada, T.Yurugi-Kobayashi, T.Shimamura, T.Asakura, K.Masuda, M.Ishiguro, T.Misaka, S.Iwata, T.Kobayashi, K.Abe.
  • Journal Title: Biophys. Acta. 1800 Pages: 986-992
  • Peer Reviewed
• [Journal Article] Characterization of the β-D-glucopyranoside binding site of the human bitter taste receptor hTAS2R16. (2010)
  • Author(s): T.Sakurai, T.Misaka, M.Ishiguro, K.Masuda, T.Sugawara, K.Ito, T.Kobayashi, S.Matsuo, Y.Ishimaru, T.Asakukra, K.Abe.
  • Journal Title: J.Biol. Chem. 285 Pages: 28373-28378
  • Peer Reviewed
• [Journal Article] Fluorescence-based optimization of human bitter taste receptor expression in Saccharomyces cerevisiae. (2009)
  • Author(s): T.Sugawara, K.Ito, M.Shiroishi, N.Tokuda, H.Asada, T.Yurugi-Kobayashi, T.Shimamura, T.Misaka, N.Nomura, T.Murata, K.Abe, S.Iwata, T.Kobayashi.
  • Journal Title: Biochem. Biophys. Res.Commun. 382 Pages: 704-710
  • Peer Reviewed
• [Journal Article] Comparison of functional non-glycosylated GPCRs expression in Pichia pastoris. (2009)
  • Author(s): T.Yurugi-Kobayashi, H.Asada, M.Shiroishi, T.Shimamura, S.Funamoto, N.Katsuta, K.Ito, T.Sugawara, N.Tokuda, H.Tsujimoto, T.Murata, N.Nomura, K.Haga, T.Haga, S.Iwata, T.Kobayashi.
  • Journal Title: Biochem. Biophys. Res.Commun. 380 Pages: 271-276
  • Peer Reviewed
• [Journal Article] Advanced method for high-throughput expression of mutated eukaryotic membrane proteins in Saccharomyces cerevisiae. (2008)
  • Author(s): K.Ito, T.Sugawara, M.Shiroishi, N.Tokuda, A.Kurokawa, T.Misaka, H.Makyio, T.Yurugi-Kobayashi, T.Shimamura, N.Nomura, T.Murata, K.Abe, S.Iwata, T.Kobayashi.
  • Journal Title: Biochem. Biophys. Res.Commun. 371 Pages: 841-845
  • Peer Reviewed
• [Journal Article] Interaction and stoichiometry of the peripheral stalk subunits NtpE and NtpF and the N-terminal hydrophilic domain of NtpI of Enterococcus hirae V-ATPase. (2008)
  • Author(s): M.Yamamoto, S.Unzai, S.Saijo, K.Ito, K.Mizutani, C.Suno-Ikeda, Y.Yabuki-Miyata, T.Terada, M.Toyama, M.Shirouzu, T.Kobayashi, Y.Kakinuma, I.Yamato, S.Yokoyama, S.Iwata, T.Murata.
  • Journal Title: J.Biol.Chem. 283 Pages: 19422-19431
  • Peer Reviewed
• [Presentation] Gタンパク共役受容体(GPCR)の三次元結晶構造解析への試み (2011)
  • Author(s): 小林拓也
  • Organizer: 第84回日本薬理学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2011-03-24
• [Presentation] A GFP-based platform for rapid construction and evaluation of highly expressed and stable GPCR variants in Saccharomyces cerevisiae (2010)
  • Author(s): 小林拓也
  • Organizer: GPCR workshop
  • Place of Presentation: Hawaii
  • Year and Date: 2010-12-10
• [Presentation] G蛋白質共役受容体(GPCR)の構造解析に向けて「生産から結晶構造解析までの技術開発」 (2009)
  • Author(s): 小林拓也
  • Organizer: 第82回日本生化学会
  • Place of Presentation: 神戸
  • Year and Date: 2009-10-22
• [Presentation] Towards Structure Determination of Human Membrane Receptors and Production of Functional GPCRs in Pichi pastoris (2008)
  • Author(s): 小林拓也
  • Organizer: 第5回GPCR研究会
  • Place of Presentation: 東京
  • Year and Date: 2008-05-09
• [Presentation] Towards Structure Determination of Human Membrane Receptors -from Prostanoid receptor studies-、播磨放射光施設 (2008)
  • Author(s): 小林拓也
  • Organizer: GPCRシンポジウム
  • Year and Date: 2008-02-26
• [Book] Gタンパク質共役受容体(GPCR)の三次元結晶構造解析への試み、23-29 (2011)
  • Author(s): 小林拓也
  • Publisher: バイオインダストリー(シーエムシー出版)
• [Book] Advanced technologies to determine the 3D structure of taste receptors. The Sense of Taste. (2011)
  • Author(s): K.Ito, T.Misaka, K.Abe, T.Kobayashi
  • Publisher: Nova Science Publishers, Inc.(印刷中)
• [Remarks] ホームページ等
  • URL: http://www.med.kyoto-u.ac.jp/J/grad_school/introduction/1502/
• [Patent(Industrial Property Rights)] 膜蛋白質の立体構造を認識するモノクローナル抗体のスクリーニング方法 (2010)
  • Inventor(s): 小林拓也、岩田想, ら
  • Industrial Property Rights Holder: 科学技術振興機構JST
  • Industrial Property Number: PCT国際出願,PCT/JP2010/57631
  • Filing Date: 20100400