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Crystallographic Studies on Protease ー Substrate Interaction Using Genetically - Engineered Inhibitors.

Research Project

Project/Area Number 01480516
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field 結晶学
Research InstitutionNagaoka University of Technology

Principal Investigator

MITSUI Yukio  Faculty of Engineering, Nagaoka University of Technology , Professor, 工学部, 教授 (40012637)

Co-Investigator(Kenkyū-buntansha) KUMAGAI Izumi  Faculty of Engineering , University of Tokyo , Assoc. Professor, 工学部, 助教授 (10161689)
Project Period (FY) 1989 – 1990
Project Status Completed (Fiscal Year 1990)
Budget Amount *help
¥4,500,000 (Direct Cost: ¥4,500,000)
Fiscal Year 1990: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1989: ¥3,400,000 (Direct Cost: ¥3,400,000)
KeywordsProtease / Protease inhibitor / Protein-protein interaction / Protein engineering / X-ray structure analysis / Crystal structure / Three-dimensional structure of Protein / 蛋白質の立体構造
Research Abstract

Crystal structure of free SSI (Streptomyces Subtilisin Inhibitor) and that of SSI complexed with its target enzyme subtilisin BPN' were refined to R-factors of 24% (1.85 A) and 21% (1.8 A) respectively. Furthermore two more related structures were refined in which subtilisin BPN' was complexed with either mutant SSI-1 or mutant SSI-2. In both mutant SSI's, the P1 residue of the inhibitor. Met 73, was converted to Lys through gene manipulation. Additionally, in mutant SSI-2 the P4 residue, Met 70, was converted to Gly.
Comparative examinations of the structural and "thermal" parameters of these structures indicate the following.
(A) Marked rigidification of the SSI structure occurs to some (but not all) parts of SSI upon complex formation with the target enzyme (subtilisin BPN').
(B) The rigidification occurs not only to the "reactive site" segment which is in direct contact with the target enzyme but also to those polypeptide segments which are simply connected to the reactive site through either covalent linkage or van der Waals contacts.
(C) The rigidified polypeptide segments of SSI mentioned above roughly correspond to the segments where marked shifts upon complex formation were observed in C-13 NMR spectra (observed by M.Kainosho and his colleague using various C-13 enriched SSI's).
(D) Basic shape of the SI pocket of subtilisin BPN' seems to be almost fixed irrespective of the nature of its "guest" (P1 residue of the inhibitor).
(E) In contrast, the S4 pocket seems to have intrinsic capacity for induced fit : it is considerably narrowed when its guests, P4 residue has smaller sidechain.

Report

(3 results)
  • 1990 Annual Research Report   Final Research Report Summary
  • 1989 Annual Research Report
  • Research Products

    (18 results)

All Other

All Publications (18 results)

  • [Publications] Y.Takeuchi,Y.Mitsui: "Structural and Functional Aspects of ProteinーProtein Interaction" GBF Monogrphs,Vol.12 ″Advances in Protein Design″. 177-183 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Y.Takeuchi,S.Kojima,K.Miura,Y.Mitsui: "Molecular Recognition at the Interfce between Subtilisim and SSI" Protein Engineering(M.Ikehra ed.)Japan Scientifac Societies Press. 103-108 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Y.Takeuchi,S.KojiMa,K.Miura,Y.Mitsui: "Molecular Recognition at the Active Site of Subtilisin BPN′" Protein Engineering. (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Y.Takuchi,K.T.Nakamura,Y.Mitsui: "Refined Crystal Structure of the Complex of Subtilisim and SSI" J.Mol.Biol.(1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] 三井 幸雄,石川 弘紀,竹内 康雄: "タンパク質の立体構造と機能" 現代化学,増刊19「プロテイン・エンジニアリング」. 145-160 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] 熊谷 泉,竹内 康雄,三井 幸雄,三浦 謹一郎: "プロテア-ゼとプロテア-ゼ・インヒビタ-" シリ-ズ 分子生物学の進歩 3.蛋白質の機能構造. 219-241 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Y. Takeuchi, Y. Mitsui et al.: "Structural and functional aspects of protein-protein interaction." Advances in Protein Design. 177-183 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Y. Takeuchi, S. Kojima, K. Miwa, Y. Mitsui et al.: "Molecular recognition at the interface between subtilisin and SSI." Protein Engineering. 103-108 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Y. Takeuchi , S. Kojima , K. Miwa . Y. Mitsui et al.: "Molecular recognition at the active site of subtilisin BPN'" Protein Engineering.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Y. Takeuchi , K. T. Nakamura , Y. Mitsui et al.: "Refined crystal structure of the complex of subtilisin and SSI" J. Mol. Biol.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Y.Takeuchi,S.Kojima,K.Miura,Y.Mitsui: "Molecular Recognition at the Interface between Subtilisin and SSI" Protein Engineering (M.Ikehara ed.)Japan Scientific Societies Press. 103-108 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Y.Takeuchi,S.Kojima,K.Miura,Y.Mitsui: "Molecular Recognition at the Active Site of Subtilisin BPN'" Protein Engineering. (1991)

    • Related Report
      1990 Annual Research Report
  • [Publications] Y.Takeuchi,K.T.Nakamura,Y.Mitsui: "Refined Crystal structure of the Complex of Subtilisim and SSI" J.Mol.Biol.(1991)

    • Related Report
      1990 Annual Research Report
  • [Publications] 三井 幸雄,石川 弘紀,竹内 康雄: "タンパク質の立体構造と機能" 現代化学,増刊19「プロティン、エンジニアリング」. 145-160 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] 熊谷 泉,竹内 康雄,三井 幸雄,三浦 謹一郎: "プロテア-ゼとプロテア-ゼ・インヒビタ-" シリ-ズ分子生物学の進歩 3.蛋白質の機能構造. 219-241 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Y.Takeuhi,Y.Mitsui: "Structral and Fuuctional Aspects of Protein-Protein Interaction" GBF Monographs,Vol.12“Advances in Protein Desigu". 12. 177-183 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Y.Takeuhi,S.Kojima,K.Miura,Y.Mitsui: "Molecular Recognition at the Interface between Subtilisin and SSI" Proc.Second International Conference on Protein Engineering. in press. (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] 熊谷泉,竹内康雄,三井幸雄,三浦謹一郎: "プロテア-ゼとプロテア-ゼ・インヒビタ-" シリ-ズ分子生物学の進歩 3.蛋白質の機能構造. 219-241 (1990)

    • Related Report
      1989 Annual Research Report

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Published: 1989-04-01   Modified: 2016-04-21  

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