Cofilin, a member of the actin-depolymerizing factor (ADF)/cofilin family of proteins, is akey regulator of actin dynamics. Cofilin binds to monomer (G-) and filamentous (F-) actin, severs thefilaments, and increases their turnover rate. Electron microscopy studies suggested cofilin interactionswith subdomains 2 and 1/3 on adjacent actin protomers in F-actin. To probe for the presence of a crypticcofilin binding site in subdomain 2 in G-actin, we used transglutaminase-mediated cross-linking, whichtargets Gln
41 in subdomain 2. The cross-linking proceeded with up to 85% efficiency with skeletal
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-actinand WT yeast actin, yielding a single product corresponding to a 1:1 actin-cofilin complex but wasstrongly inhibited in Q41C yeast actin (in which Q41 was substituted with cysteine). LC-MS/MS analysisof the proteolytic fragments of this complex mapped the cross-linking to Gln
41 on actin and Gly
1 onrecombinant yeast cofilin. The actin-cofilin (AC) heterodimer was purified on FPLC for analyticalultracentrifugation and electron microscopy analysis. Sedimentation equilibrium and velocity runs revealedoligomers of AC in G-actin buffer. In the presence of excess cofilin, the covalent AC heterodimer bounda second cofilin, forming a 2:1 cofilin/actin complex, as revealed by sedimentation results. Underpolymerizing conditions the cross-linked AC formed mostly short filaments, which according to imagereconstruction were similar to uncross-linked actin-cofilin filaments. Although a majority of the cross-linking occurs at Gln
41, a small fraction of the AC cross-linked complex forms in the Q41C yeast actinmutant. This secondary cross-linking site was sequenced by MALDI-MS/MS as linking Gln
360 in actinto Lys
98 on cofilin. Overall, these results demonstrate that the region around Gln
41 (subdomain 2) is involvedin a weak binding of cofilin to G-actin.