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

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.

Pubmed ID: 18258262


  • Grintsevich EE
  • Benchaar SA
  • Warshaviak D
  • Boontheung P
  • Halgand F
  • Whitelegge JP
  • Faull KF
  • Loo RR
  • Sept D
  • Loo JA
  • Reisler E


Journal of molecular biology

Publication Data

March 21, 2008

Associated Grants

  • Agency: NIGMS NIH HHS, Id: GM 077190
  • Agency: NIGMS NIH HHS, Id: GM-067246
  • Agency: NIGMS NIH HHS, Id: R01 GM067246
  • Agency: NIGMS NIH HHS, Id: R01 GM067246-04
  • Agency: NIGMS NIH HHS, Id: R01 GM077190
  • Agency: NIGMS NIH HHS, Id: R01 GM077190-31
  • Agency: NCRR NIH HHS, Id: R01 RR020004
  • Agency: NCRR NIH HHS, Id: R01 RR020004-04
  • Agency: NCRR NIH HHS, Id: RR 20004

Mesh Terms

  • Actin Depolymerizing Factors
  • Actins
  • Amino Acid Sequence
  • Binding Sites
  • Cross-Linking Reagents
  • Ethylmaleimide
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Protein Binding
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae
  • Structural Homology, Protein