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 α-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 ultrace...