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

http://www.ncbi.nlm.nih.gov/pubmed/18258262

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 RIS Download

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

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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

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