Cofilin Binding to Globular and Filamentous Actin

Abstract

Cofilin is important for the regulation of the actin cytoskeleton. It is capable of binding to and severing F-actin filaments, but the molecular basis for these functions is poorly understood, due to a paucity of structural data on the quaternary complex. A recent computational model has been proposed (Galkin et al., 2011, PNAS, 108:20568). To test this model, we inserted mutant residues into the sequence of cofilin to facilitate specific labelling with spectroscopic probes. We were able to successfully mutate and express four mutant cofilins. We were hampered in this process by the apparent sensitivity of the structure of cofilin to mutagenesis. Thus, we found that mutagenesis of the N-terminus abolished G-actin binding, while still permitting F actin binding, reinforced the importance of the N-terminus of cofilin in binding to actin. On the other hand, mutation of the C-terminus of cofilin and W135 had no effect on the binding of cofilin to G-actin. Mutation of W104 abolished G-actin binding, highlights the importance of the β5 strand and α6 helix of cofilin in actin binding via the G/F actin binding site on cofilin. Fortunately, the mutant forms of cofilin that retained binding to G actin (Cys170 cofilin and Cys170 W104 cofilin) retained their capacity to bind to G-actin after modification with extrinsic spectroscopic probes, allowing us to measure one distance within cofilin and three distances between cofilin and G-actin. The distances between cofilin and G-actin corresponded closely to the predicted distances from the Galkin model.