Crystallization of GPCR and its specific antibody

2012 Fiscal Year Final Research Report

• Project/Area Number: 22590270
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: General medical chemistry
• Research Institution: Kyoto University
• Principal Investigator: ASADA Hidetsugu 京都大学, 大学院・医学研究科, 特定助教 (20399041)
• Co-Investigator(Kenkyū-buntansha): SHIROISHI Mitsunori 九州大学, 大学院・薬学研究科, 助教 (00380527)
• Project Period (FY): 2010 – 2012
• Keywords: 構造生物 / GPCR / 抗体 / 結晶化 / X線結晶構造解析

Research Abstract

Angiotensin II receptor type2 (AT2) play an importantrole for regulating blood pressure. Recent studies strongly suggest that AT2 not only mediates blood pressure control but also plays an important role in cardiovascular protection such as anti cardiac hypertrophy. To solve AT2 structure is important for understanding of the molecular mechanism of blood pressure control and developing of drug against cardiac hypertrophy. Recently, several GPCR structures have been solved. However, to determine of GPCR crystal structure still remains having some problems. In this study, we developed AT2 structure specific recognition antibodies to overcome these problems. We have been trying to crystallize of AT2 with Fab in order to solve the structure in this study. Stabilized AT2 mutant (AT2-T4L) has been constructed by deletion of the flexible N- and C-terminus and insertion of T4 lysozyme (T4L) to the third intracellular loop (i3). AT2-T4L has been expressed in Sf9 insect cells. The expression lev els of AT2-T4L have been assessed by flow cytometric analysis (FACS) and specific ligand-binding assay. We have purified from the cell membrane expressing AT2-T4L using TALON resin and size exclusion chromatography (SEC). The characteristics of the purified receptors have been assessed by thermal stability (CPM) assay. The antibodies have been developed from immunization of mice with wild type AT2. The antibodies have been screened using liposome-ELISA, dot plot analysis, FACS, and fluorescence size exclusion chromatography (FSEC). We have prepared Fab region from the screened antibodies for crystallization. The purified AT2T4L has been incubated with its Fab. Then, the complex has been purified by means of SEC. The complex has been crystallized in vapordiffusion. The AT2-T4L could be expressed and purified successfully. The expression level was 57.3±8.9% (Average±SD) of whole infected cells (FACS) and 91.7±37.8pmol/mg -membrane (ligand-binding assay). The thermal stability of AT2-T4L had melting temperature (Tm) of 34 and 58℃. This data show that thermal stability of AT2-T4L is not enough for crystallization. Actually, we could not acquire the crystals from this sample in LCP. To contribute improvement of AT2-T4L stability, we have established three AT2 specific antibodies via the screening steps. The complex of AT2-T4L and Fab had Tm of 62℃. This thermal stability was higher than AT2-T4L. Furthermore, the thermal stability of complex was maintained for a week at 4℃. These data strongly suggested the complex is more suitable for crystallization. The complex was crystallized in vapor diffusion. We acquired some crystals of complex but their resolution had very low at the present study. So, we have been trying to crystallization in vapor diffusion and LCP under the various conditions.

Research Products

• [Journal Article] Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist (2012)
  • Author(s): Haga, K*., A. C. Kruse*,H. Asada*, T. Yurugi-Kobayashi, M. Shiroishi, C. Zhang, W. I. Weis, T. Okada, B. K. Kobilka, T. Haga, and T. Kobayashi
  • Journal Title: These authors contributed equally to this work Volume: 482 Pages: 547-551
• [Journal Article] Production of the stable human histamine H1 receptor in Pichia pastoris for structural determination (2011)
  • Author(s): Shiroishi, M, T. Kobayashi, S. Ogasawara, H. Tsujimoto, C. Ikeda-Suno, S. Iwata, T. Shimamura
  • Journal Title: Methods Volume: 55 Pages: 281-286
• [Journal Article] Crystal structure-based virtual screening for fragment-like ligands of the human histamine H(1) receptor (2011)
  • Author(s): de Graaf C, AJ. Kooistra, HF. Vischer, V. Katritch, M. Kuijer, M. Shiroishi, S. Iwata, T. Shimamura, RC. Stevens, IJ. de Esch, R. Leurs
  • Journal Title: J Med Chem Volume: 54 Pages: 8195-8206
• [Journal Article] Molecular Basis for Herpesvirus Entry Mediator Recognition by the Human Immune Inhibitory Receptor CD160 and Its Relationship to the Cosignaling Molecules BTLA and LIGHT (2011)
  • Author(s): Kojima R, M. Kajikawa, M. Shiroishi, K. Kuroki, K. Maenaka
  • Journal Title: J Mol Biol Volume: 413 Pages: 762-772
• [Journal Article] Structure of the human histamine H(1) receptor complex with doxepin, These authors contributed equally to this work (2011)
  • Author(s): Shimamura T*, M. Shiroishi *, S. Weyand, H. Tsujimoto, G. Winter, V. Katritch, R. Abagyan, V. Cherezov, W. Liu, GW. Han, T. Kobayashi, RC. Stevens, S. Iwata
  • Journal Title: Nature Volume: 475 Pages: 65-70
• [Journal Article] Cloning, expression and purification of the anion exchanger 1 homologue from the basidiomycete Phanerochaete chrysosporium (2011)
  • Author(s): Tokuda, N., K. Igarashi, T. Shimamura, T. Yurugi-Kobayashi, M. Shiroishi, K. Ito, T. Sugawara, H. Asada, T. Murata, N. Nomura, S. Iwata, and T. Kobayashi
  • Journal Title: Protein Expr Purif Volume: 79 Pages: 81-87
• [Journal Article] Cysteine-to-serine shuffling using a Saccharomyces cerevisiae expression system improves protein secretion: case of a nonglycosylated mutant of miraculin, a taste-modifying protein (2011)
  • Author(s): Ito, K., T. Sugawara, A. Koizumi, K.Nakajima, A. Shimizu-Ibuka, M. Shiroishi, H. Asada, T. Yurugi-Kobayashi, T. Shimamura, T. Asakura, T. Misaka, S. Iwata, T. Kobayashi, and K. Abe
  • Journal Title: Biotechnol Lett Volume: 33 Pages: 103-107
• [Journal Article] Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis (2011)
  • Author(s): Asada, H., 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, and T. Kobayashi
  • Journal Title: Microb Cell Fact Volume: 10 Pages: 24
• [Journal Article] Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin (2010)
  • Author(s): Ito, K., T. Sugawara, A. Koizumi, K. 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, and K. Abe
  • Journal Title: Biochim Biophys Acta Volume: 1800 Pages: 986-992
• [Journal Article] Contribution of asparagine residues to the stabilization of a proteinaceous antigen-antibody complex, HyHEL-10-hen egg white lysozyme (2010)
  • Author(s): Yokota A, K. Tsumoto, M. Shiroishi, T. Nakanishi, H. Kondo, I. Kumagai
  • Journal Title: J Biol Chem Volume: 285 Pages: 7686-96