Mapping the cofilin binding site on yeast G-actin by chemical cross-linking. - CNRS - Centre national de la recherche scientifique Accéder directement au contenu
Article Dans Une Revue Journal of Molecular Biology Année : 2008

Mapping the cofilin binding site on yeast G-actin by chemical cross-linking.

Elena E Grintsevich
  • Fonction : Auteur
Sabrina A Benchaar
  • Fonction : Auteur
Dora Warshaviak
  • Fonction : Auteur
Pinmanee Boontheung
  • Fonction : Auteur
Julian P Whitelegge
  • Fonction : Auteur
Kym F Faull
  • Fonction : Auteur
Rachel R Ogorzalek Loo
  • Fonction : Auteur
David Sept
  • Fonction : Auteur
Joseph A Loo
  • Fonction : Auteur
Emil Reisler
  • Fonction : Auteur

Résumé

Cofilin is a major cytoskeletal protein that binds to both monomeric actin (G-actin) and polymeric actin (F-actin) and is involved in microfilament dynamics. Although an atomic structure of the G-actin-cofilin complex does not exist, models of the complex have been built using molecular dynamics simulations, structural homology considerations, and synchrotron radiolytic footprinting data. The hydrophobic cleft between actin subdomains 1 and 3 and, alternatively, the cleft between actin subdomains 1 and 2 have been proposed as possible high-affinity cofilin binding sites. In this study, the proposed binding of cofilin to the subdomain 1/subdomain 3 region on G-actin has been probed using site-directed mutagenesis, fluorescence labeling, and chemical cross-linking, with yeast actin mutants containing single reactive cysteines in the actin hydrophobic cleft and with cofilin mutants carrying reactive cysteines in the regions predicted to bind to G-actin. Mass spectrometry analysis of the cross-linked complex revealed that cysteine 345 in subdomain 1 of mutant G-actin was cross-linked to native cysteine 62 on cofilin. A cofilin mutant that carried a cysteine substitution in the alpha 3-helix (residue 95) formed a cross-link with residue 144 in actin subdomain 3. Distance constraints imposed by these cross-links provide experimental evidence for cofilin binding between actin subdomains 1 and 3 and fit a corresponding docking-based structure of the complex. The cross-linking of the N-terminal region of recombinant yeast cofilin to actin residues 346 and 374 with dithio-bis-maleimidoethane (12.4 A) and via disulfide bond formation was also documented. This set of cross-linking data confirms the important role of the N-terminal segment of cofilin in interactions with G-actin.

Dates et versions

hal-00275501 , version 1 (24-04-2008)

Identifiants

Citer

Elena E Grintsevich, Sabrina A Benchaar, Dora Warshaviak, Pinmanee Boontheung, Frédéric Halgand, et al.. Mapping the cofilin binding site on yeast G-actin by chemical cross-linking.. Journal of Molecular Biology, 2008, 377 (2), pp.395-409. ⟨10.1016/j.jmb.2007.12.073⟩. ⟨hal-00275501⟩
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