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.
Grintsevich
Elena E
EE
Department of Chemistry and Biochemistry, University of California-Los Angeles, CA, USA.
Benchaar
Sabrina A
SA
Warshaviak
Dora
D
Boontheung
Pinmanee
P
Halgand
Frédéric
F
Whitelegge
Julian P
JP
Faull
Kym F
KF
Loo
Rachel R Ogorzalek
RR
Sept
David
D
Loo
Joseph A
JA
Reisler
Emil
E
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
D051339:Actin Depolymerizing Factors; D000199:Actins; D003432:Cross-Linking Reagents; C021017:dithiobis(N-ethylmaleimide); D005033:Ethylmaleimide