The second ADF/cofilin actin‐binding site exists in F‐actin, the cofilin–G‐actin complex, but not in G‐actin

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Abstract
ADF/cofilins are actin binding proteins that bind actin close to both the N‐ and C‐termini (site 1), and we have found a second cofilin binding site (site 2) centered around helix 112–125 [Renoult, C., Ternent, D., Maciver, S.K., Fattoum, A., Astier, C., Benyamin, Y. & Roustan, C. (1999) J. Biol. Chem.274, 28893–28899]. We proposed a model in which ADF/cofilin intercalated between subdomains 1 and 2 of two longitudinally associated actin monomers within the actin:cofilin cofilament, explaining the change in twist that ADF/cofilins induce in the filament [McGough, A. Pope, B., Chiu, W. & Weeds, A. (1998) J. Cell Biol.138, 771–781]. Here, we have determined the fuller extent of the cofilin footprint on site 1 of actin. Site 1 is primarily the G‐actin binding site. Experiments with both peptide mimetics and fluorescently labeled cofilin suggest that site 2 only becomes available for cofilin binding within the filament, possibly due to motion between subdomains 1 and 2 within an actin monomer. We have detected motion between subdomains 1 and 2 of G‐actin by FRET induced by cofilin, to reveal the second cofilin‐binding site. This motion may also explain how cofilins inhibit the nucleotide exchange of actin, and why the actin:cofilin complex is polymerizable without dissociation.