Etude du rôle du zinc et des cystéines dans la dimérisation de la protéine FUR (Ferric Uptake Regulator) d'E. Coli : une approche structurale par RMN

par Ludovic Pecqueur

Thèse de doctorat en Biologie structurale et fonctionnelle

Sous la direction de Beate Bersch et de Isabelle Michaud-Soret.

Soutenue en 2005

à l'Université Joseph Fourier (Grenoble) .

    mots clés mots clés


  • Résumé

    La protéine FUR (Ferric Uptake Regulator) est un régulateur global ubiquitaire chez les bactéries Gram-négatives. Sa liaison au Fe2+, in vivo, entraîne la répression de l'expression des gènes qu'elle contrôle. Ce travail est une étude structurale par RMN de la forme dimérique non activée de FUR d'Escherichia coli, un dimère de 2*17 kDa contenant un ion zinc par monomère. Une forme monomérique oxydée, capable de dimériser en présence de réducteur et de zinc, a également été isolée et e��tudiée. Le dichroïsme circulaire et la RMN montrent que la dimérisation entraîne une structuration du domaine C-terminal lors de l'incorporation du zinc. Les structures secondaires du domaine N-terminal du monomère et du dimère sont très proches. Seuls les premiers résidus sont structurés en hélice α dans le monomère et déstructurés dans le dimère non activé. Cette hélice, observée dans le dimère activé de FUR de P. Aeruginosa, pourrait jouer un rôle dans le mécanisme de régulation. Une protéine tronquée (FUR1-82) a été construite, purifiée, cristallisée. Sa structure est superposable à celle du domaine N-terminal de FUR de P. Aeruginosa et le spectre 1H-15N-HSQC est superposable aux signaux du domaine N-terminal de FUR monomère. L'étude, par anisotropie de fluorescence, de la liaison du monomère et du dimère à l'ADN montre qu'ils se lient spécifiquement à l'ADN en présence de métal, contrairement à la forme tronquée. L'affinité du monomère pour l'ADN est 5 fois plus faible que celle du dimère en excès de métal. L'ensemble de ces données nous a permis de proposer un mécanisme de dimérisation de FUR d'E. Coli ainsi qu'un mécanisme d'activation mettant en jeu cette hélice α N-terminale.


  • Pas de résumé disponible.

  • Titre traduit

    Role of the zinc and the cysteines in the dimerization of the Ferric Uptake Regulator (FUR) from E. Coli : a structural approach using NMR


  • Résumé

    The FUR protein (Ferric Uptake Regulator) is a global regulator ubiquitous in Gram-negative bacteria. Binding of Fe2+ in vivo activates the FUR protein for DNA binding. Fe-FUR binds to the promotor of FUR regulated genes, causing their repression. This work consists of an NMR structural study of the non-activated dimer of FUR (2*17 kDa) which contains one zinc ion per monomer. The FUR monomer was also studied and does not contain the zinc ion. Reduction of the cysteines, and binding of zinc to the protein, leads to dimerization. Circular dichroism and NMR showed that folding of the C-terminal domain of the monomer (dimerization domain) occurs upon dimerization. The secondary structure observed in the monomer and the dimer are similar, except for the first residues. In the monomer, these are organized as a helix while they are unfolded in the non-activated dimer. As this first helix is observed in the activated dimer of P. Aeruginosa, we propose it plays a role in the regulation mechanism. A truncated protein named FUR1-82 was constructed, purified, and crystallized. Its structure is superimposable onto the N-terminal domain structure of P. Aeruginosa FUR and the 1H-15N-HSQC spectra of FUR1-82 and FUR monomer are nearly identical. Binding of the monomer and the dimer to the DNA consensus sequence study, using fluorescence anisotropy, showed that both forms are able to bind DNA specifically, but with different affinities. The monomer has a 5 fold decreased affinity compared to the dimer in presence of excess metal. Overall, the results obtained allowed us to propose a dimerization mechanism of E. Coli FUR and an activation mechanism with a role for the N-terminal The FUR protein (Ferric Uptake Regulator) is a global regulator ubiquitous in Gram-negative bacteria. Binding of Fe2+ in vivo activates the FUR protein for DNA binding. Fe-FUR binds to the promotor of FUR regulated genes, causing their repression. This work consists of an NMR structural study of the non-activated dimer of FUR (2*17 kDa) which contains one zinc ion per monomer. The FUR monomer was also studied and does not contain the zinc ion. Reduction of the cysteines, and binding of zinc to the protein, leads to dimerization. Circular dichroism and NMR showed that folding of the C-terminal domain of the monomer (dimerization domain) occurs upon dimerization. The secondary structure observed in the monomer and the dimer are similar, except for the first residues. In the monomer, these are organized as a helix while they are unfolded in the non-activated dimer. As this first helix is observed in the activated dimer of P. Aeruginosa, we propose it plays a role in the regulation mechanism. A truncated protein named FUR1-82 was constructed, purified, and crystallized. Its structure is superimposable onto the N-terminal domain structure of P. Aeruginosa FUR and the 1H-15N-HSQC spectra of FUR1-82 and FUR monomer are nearly identical. Binding of the monomer and the dimer to the DNA consensus sequence study, using fluorescence anisotropy, showed that both forms are able to bind DNA specifically, but with different affinities. The monomer has a 5 fold decreased affinity compared to the dimer in presence of excess metal. Overall, the results obtained allowed us to propose a dimerization mechanism of E. Coli FUR and an activation mechanism with a role for the N-terminal The FUR protein (Ferric Uptake Regulator) is a global regulator ubiquitous in Gram-negative bacteria. Binding of Fe2+ in vivo activates the FUR protein for DNA binding. Fe-FUR binds to the promotor of FUR regulated genes, causing their repression. This work consists of an NMR structural study of the non-activated dimer of FUR (2*17 kDa) which contains one zinc ion per monomer. The FUR monomer was also studied and does not contain the zinc ion. Reduction of the cysteines, and binding of zinc to the protein, leads to dimerization. Circular dichroism and NMR showed that folding of the C-terminal domain of the monomer (dimerization domain) occurs upon dimerization. The secondary structure observed in the monomer and the dimer are similar, except for the first residues. In the monomer, these are organized as a helix while they are unfolded in the non-activated dimer. As this first helix is observed in the activated dimer of P. Aeruginosa, we propose it plays a role in the regulation mechanism. A truncated protein named FUR1-82 was constructed, purified, and crystallized. Its structure is superimposable onto the N-terminal domain structure of P. Aeruginosa FUR and the 1H-15N-HSQC spectra of FUR1-82 and FUR monomer are nearly identical. Binding of the monomer and the dimer to the DNA consensus sequence study, using fluorescence anisotropy, showed that both forms are able to bind DNA specifically, but with different affinities. The monomer has a 5 fold decreased affinity compared to the dimer in presence of excess metal. Overall, the results obtained allowed us to propose a dimerization mechanism of E. Coli FUR and an activation mechanism with a role for the N-terminal α helix.

Consulter en bibliothèque

La version de soutenance existe sous forme papier

Informations

  • Détails : 1 vol. (290 p.)
  • Notes : Publication autorisée par le jury
  • Annexes : Bibliogr. en fin de chapitres

Où se trouve cette thèse ?

  • Bibliothèque : Service interétablissements de Documentation (Saint-Martin d'Hères, Isère). Bibliothèque universitaire de Sciences.
  • Non disponible pour le PEB
  • Cote : TS05/GRE1/0265
  • Bibliothèque : Service interétablissements de Documentation (Saint-Martin d'Hères, Isère). Bibliothèque universitaire de Sciences.
  • Disponible pour le PEB
  • Cote : TS05/GRE1/0265/D
Voir dans le Sudoc, catalogue collectif des bibliothèques de l'enseignement supérieur et de la recherche.