title: Mapping the cofilin binding site on yeast G-actin by chemical cross-linking.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California-Los Angeles, CA, USA.
authors.forename: Elena E
authors.initials: EE
authors.lastname: Grintsevich
authors.forename: Sabrina A
authors.initials: SA
authors.lastname: Benchaar
authors.forename: Dora
authors.initials: D
authors.lastname: Warshaviak
authors.forename: Pinmanee
authors.initials: P
authors.lastname: Boontheung
authors.forename: Frédéric
authors.initials: F
authors.lastname: Halgand
authors.forename: Julian P
authors.initials: JP
authors.lastname: Whitelegge
authors.forename: Kym F
authors.initials: KF
authors.lastname: Faull
authors.forename: Rachel R Ogorzalek
authors.initials: RR
authors.lastname: Loo
authors.forename: David
authors.initials: D
authors.lastname: Sept
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Loo
authors.forename: Emil
authors.initials: E
authors.lastname: Reisler
mesh_terms: D051339:Actin Depolymerizing Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000199:Actins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry*; D005033:Ethylmaleimide / Q000031:analogs & derivatives / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein
chemical_list: D051339:Actin Depolymerizing Factors; D000199:Actins; D003432:Cross-Linking Reagents; C021017:dithiobis(N-ethylmaleimide); D005033:Ethylmaleimide