Mapping the interaction of cofilin with subdomain 2 on actin.
Cofilin, a member of the actin-depolymerizing factor (ADF)/cofilin family of proteins, is a key regulator of actin dynamics. Cofilin binds to monomer (G-) and filamentous (F-) actin, severs the filaments, and increases their turnover rate. Electron microscopy studies suggested cofilin interactions with subdomains 2 and 1/3 on adjacent actin protomers in F-actin. To probe for the presence of a cryptic cofilin binding site in subdomain 2 in G-actin, we used transglutaminase-mediated cross-linking, which targets Gln41 in subdomain 2. The cross-linking proceeded with up to 85% efficiency with skeletal alpha-actin and WT yeast actin, yielding a single product corresponding to a 1:1 actin-cofilin complex but was strongly inhibited in Q41C yeast actin (in which Q41 was substituted with cysteine). LC-MS/MS analysis of the proteolytic fragments of this complex mapped the cross-linking to Gln41 on actin and Gly1 on recombinant yeast cofilin. The actin-cofilin (AC) heterodimer was purified on FPLC for analytical ultracentrifugation and electron microscopy analysis. Sedimentation equilibrium and velocity runs revealed oligomers of AC in G-actin buffer. In the presence of excess cofilin, the covalent AC heterodimer bound a second cofilin, forming a 2:1 cofilin/actin complex, as revealed by sedimentation results. Under polymerizing conditions the cross-linked AC formed mostly short filaments, which according to image reconstruction were similar to uncross-linked actin-cofilin filaments. Although a majority of the cross-linking occurs at Gln41, a small fraction of the AC cross-linked complex forms in the Q41C yeast actin mutant. This secondary cross-linking site was sequenced by MALDI-MS/MS as linking Gln360 in actin to Lys98 on cofilin. Overall, these results demonstrate that the region around Gln41 (subdomain 2) is involved in a weak binding of cofilin to G-actin.
Benchaar
Sabrina A
SA
Department of Chemistry and Biochemistry, Molecular Biology Institute and Department of Biological Chemistry, University of California, Los Angeles, California 90095, USA.
Xie
Yongming
Y
Phillips
Martin
M
Loo
Rachel R Ogorzalek
RR
Galkin
Vitold E
VE
Orlova
Albina
A
Thevis
Mario
M
Muhlrad
Andras
A
Almo
Steven C
SC
Loo
Joseph A
JA
Egelman
Edward H
EH
Reisler
Emil
E
D051339:Actin Depolymerizing Factors / Q000737:chemistry* / Q000378:metabolism*; D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D011503:Transglutaminases / Q000378:metabolism; D014461:Ultracentrifugation; D015003:Yeasts / Q000378:metabolism
D051339:Actin Depolymerizing Factors; D000199:Actins; D003432:Cross-Linking Reagents; D005973:Glutamine; D011503:Transglutaminases