• Register
X
Forgot Password

If you have forgotten your password you can enter your email here and get a temporary password sent to your email.

X

Leaving Community

Are you sure you want to leave this community? Leaving the community will revoke any permissions you have been granted in this community.

No
Yes

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

Authors

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

Journal

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