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1 X (crystallizing ligand) Fv Fv 3.5 Co-crystallization of membrane proteins with antibody fragments may emerge as a general tool to facilitate crystal growth and improve crystal quality. The bound antibody fragment enlarges the hydrophilic part of the mostly hydrophobic membrane protein, thereby increasing the interaction area for possible protein-protein contacts in the crystal. To serve as a crystallizing ligand, the antibody fragment has to recognize an epitope that is only present in the native conformation (and not in the denatured state) of the membrane protein, bind with high affinity, and form stable and rigid complexes. In this project, we developed a fast and reliable method for generating crystallizing ligands against various mammalian G-protein coupled receptors (GPCRs) and transporters in a phage display format. Using this method, the crystal structure of a mammalian facilitative glucose transporter complexed with a Fv fragment has been determined at 3.5 resolution. X

50% SGDD: Structure Guided Drug Development 2009 10 Protein Data Bank (PDB) 60,800 250 ( 0.

2 50% SGDD: Structure Guided Drug Development Protein Data Bank (PDB) 60, ( 0.4%) (crystallizing ligand) 1 X Fab 50 kda GPCR Fab Fv V H H scfv Fv X scfv mg (1) SLC GLUT512 Na + (SGLTs) Na + (GLUTs) 14 (GLUT114) X GLUTs

3 GLUT5 (2) 2 2. nm (bait) (2) scfv lipid A RNA (V H V L ) cdna RT-PCR RT-PCR (SSGGGGSGGGGGGSSRSS) scfv DNA PCR pcomb3xss Carlos F. Barbas SfiI cfu 20 DNA (CDR) B V H V L cdna Dynabeads MyOne Streptavidin T1 (Invitrogen) 4 scfv scfv ELISA ELISA (1) GLUT5 3 4D111 4D151 4D160 scfv ELISA Biacore T-100 C (HL ) HA Biacore SA HA IgG 2 10

pny326 Sec V L cdna Brevibacillus V L V H 1:1 2530 6570 10kDa 4

4 3 Fv 4. Fv 3. Biacore (2) Pichia pastoris mg Brevibacillus choshinensis Fv Fab pny326 Sec V L cdna Brevibacillus V L V H 1: kDa 4 Fv Fv 1 L 810 mg Fv 10 mg/ml (3) GLUT5 N50Y Fv 5 5. a: b: GLUT5/Fv SPring-8 BL41XU X (outward-open conformation)fv

84 2011 9 24 1 6. GLUT5 4 Tokuda N, Igarashi K, Shimamura T, Yurugi-Kobayashi T, Shiroishi M, Ito K, Sugawara T, Asada H, Murata T, Nomura N, Iwata S, Kobayashi T.

5 GLUT5 4 Tokuda N, Igarashi K, Shimamura T, Yurugi-Kobayashi T, Shiroishi M, Ito K, Sugawara T, Asada H, Murata T, Nomura N, Iwata S, Kobayashi T. Cloning, expression and purification of the anion exchanger 1 homologue from the basidiomycete Phanerochaete chrysosporium. Protein Expr Purif Sep;79(1):81-7. DOI: /j.pep Asada H, Uemura T, Yurugi-Kobayashi T, Shiroishi M, Shimamura T, Tsujimoto H, Ito K, Sugawara T, Nakane T, Nomura N, Murata T, Haga T, Iwata S, Kobayashi T. Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis. Microb Cell Fact Apr 22;10:24. DOI: / (1) NOMURA NORIMICHI (2) (3) IWATA SO Hino T, Arakawa T, Iwanari H, Yurugi-Kobayashi T, Ikeda-Suno C, Nakada-Nakura Y, Kusano-Arai O, Weyand S, Shimamura T, Nomura N, Cameron AD, Kobayashi T, Hamakubo T, Iwata S, Murata T. G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody. Nature Jan 29;482(7384): DOI: /nature10750 Shiroishi M, Tsujimoto H, Makyio H, Asada H, Yurugi-Kobayashi T, Shimamura T, Murata T, Nomura N, Haga T, Iwata S, Kobayashi T. Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae. Microb Cell Fact Jun 13;11:78. DOI: / X

研究成果報告書

様式 C-19 科学研究費補助金研究成果報告書平成 23 年 4 月 5 日現在機関番号 :14301 研究種目 : 基盤研究 (B) 研究期間 :2008~2010 課題番号 :20370035 研究課題名 ( 和文 ) バンド 3 の三次元結晶構造解析と陰イオン透過分子機序の解明研究課題名 ( 英文 ) Crystal structure of human Band3 (anion exchanger)