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Crystallographical and molecular biological analysis of ia component of C. perfringens iota-toxin

Research Project

Project/Area Number 12670270
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Bacteriology (including Mycology)
Research InstitutionTokushima Bunri University

Principal Investigator

SAKURAI J  Tokushima Bunri Univ., Fac. Pharm. Sci. full Professor, 薬学部, 教授 (80029800)

Co-Investigator(Kenkyū-buntansha) KOBAYASHI Keiko  Tokushima Bunri Univ., Fac. Pharm. Res. Associate, 薬学部, 助手 (90170315)
OCHI Sadayuki  Tokushima Bunri Univ., Fac. Pharm. Res. Associate, 薬学部, 助手 (80268705)
NAGAHAMA Masahiro  Tokushima Bunri Univ., Fac. Pharm. Sci. Asso. Professor, 薬学部, 助教授 (40164462)
Project Period (FY) 2000 – 2001
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2001: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2000: ¥2,100,000 (Direct Cost: ¥2,100,000)
KeywordsClostridium perfringens / iotaa component / ADP-Ribosylation / NAD / crystallogaraphy / site-directed mutagenesis / ADP-ribosylatiing toxin / Sn1 type reaction / ADPリボシル化毒素 / 結晶解析 / アミノ酸置換 / カイネティク解析 / ADPリボシル化酵素 / NADディハイドロゲナーゼ活性 / ADPリボシル転移活性 / アクチン
Research Abstract

The iotaa component (ia) of Clostridium perfringens ADP-ribosylates nonmuscld (β/γ actin and skeletal muscle α-actin. Here, we showed crystal structures of Ia complex with NADH at 1.8Å. It was found that Ia can be divided into two domains, termed the N-domain and the C-domain that has a cavity which binds NADH. A site-directed mutagenesis based on the structure suggested that the catalytic mechanism of Ia proceeds via Snl type reaction as follows ; the positively charged atoms from Arg295 and Arg352 electrically interact with α- and β-phosphate groups of NAD^+, respectively, Glu380 fixes the ribose position, the benzene ring of Phe349 restricts the nicotinamide ring rotation and the nicotinamide carboxyamide forms hydrogen bonds with the main chain amide and carbonyl of Arg296.The specific binding of NAD^+ to Ia induces a conformational change in NAD^+ that results in a ring-like conformation of NAD in the complex. The α-phosphate group could be most important for cleavage via Snl-type reaction, by making a hydrogen bond with the amino group of nicotinamide (distance of 2.92Å) and then withdrawing the electron from the amino group. Thus the hydrogen bond formation induces the spontaneous cleavage of N-glycoside bond. The resulting oxocarbonium cation of the ADP-ribosyl moiety should be stabilized by chelation with the negatively charged carboxylate group of Glu378.The Arg177 of actin is located near the oxocarbonium cation upon Ia-actin complex formation. Then the ADP-ribose group is transferred to the Arg177 of actin.

Report

(3 results)
  • 2001 Annual Research Report   Final Research Report Summary
  • 2000 Annual Research Report
  • Research Products

    (9 results)

All Other

All Publications (9 results)

  • [Publications] S.Ochi, M.Miyawaki, H.Matsuda, M.Oda, M, Nagahama, J.Sakurai: "Clostridium perfringens α-toxin induces rabbit neutrophil adhesion"Microbiology. 148. 237-245 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] M.Nagahama, K.Nagayasu, K.Kobayashi, J.Sakurai: "Binding component of Clostridium perfringens iota-toxin induces endocytosis in Vero cells"Infect.Immun.. 70(In press). (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] S, Ochi.M, Miyawaki.H, Matsuda.M, Oda.M, Nagahama. And J, Sakurai: "Clostridium perfringens α-toxin induces rabbit neutrophil adhesion."Microbiology. 148. 237-245 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] M, Nagahama.K, Nagayasu.K, Kobayashi. And J, Sakurai.: "Binding component of Clostridium perfringens iota-toxin induces endocytosis in Vero cells"Infect. Immun. 70(4) (in press). (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] S.Ochi, M.Miyawaki, H.Matsuda, M.Oda, M, Nagahama, J.Sakurai: "Clostridium perfringens α-toxin induces rabbit neutrophil adhesion"Microbiology. 148. 237-245 (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] M.Nagahama, K.Nagayasu, K.Kobayashi, J.Sakurai: "Binding component of Clostridium perfringens iota-toxin induces endocytosis in Vero cells"Infection and Immunity. 70(In press). (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] 櫻井 純: "イラストレイテッド微生物学 第2版"南山堂. 372 (2000)

    • Related Report
      2001 Annual Research Report
  • [Publications] M.Nagahama,M.Mukai,S.Ochi,and J.Sakurai: "Role of tryptophan-1 in hemolytic and Phospholipase C activities of Clostridium perfringens alpha-toxin"Microbiol.Immunol.. 44・7. 585-589 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] M.Nagahama,Y.Sakaguchi,K.Kobayashi,S.Ochi,and J.Sakusa: "Characterization of the enzymatic component of Clostridium perfringens Iota-toxin."J.Bacteriol.. 182・8. 2096-2103 (2000)

    • Related Report
      2000 Annual Research Report

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Published: 2000-04-01   Modified: 2016-04-21  

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