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Antagonistic Effects of Cofilin, Beryllium Fluoride Complex, and Phalloidin on Subdomain 2 and Nucleotide-Binding Cleft in F-Actin

Andras Muhlrad ^*, Israel Ringel , Dmitry Pavlov , Y. Michael Peyser ^* and Emil Reisler 

^* Institute of Dental Sciences, School of Dental Medicine, Department of Pharmacology, School of Pharmacy, Hebrew University of Jerusalem, Jerusalem, Israel; and Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, California

Correspondence: Address reprint requests to Andras Muhlrad, Fax: 972-2-675-8561; E-mail: muhlrad{at}cc.huji.ac.il.

Cofilin/ADF, beryllium fluoride complex (BeFx), and phalloidin have opposing effects on actin filament structure and dynamics. Cofilin/ADF decreases the stability of F-actin by enhancing disorder in subdomain 2, and by severing and accelerating the depolymerization of the filament. BeFx and phalloidin stabilize the subdomain 2 structure and decrease the critical concentration of actin, slowing the dissociation of monomers. Yeast cofilin, unlike some other members of the cofilin/ADF family, binds to F-actin in the presence of BeFx; however, the rate of its binding is strongly inhibited by BeFx and decreases with increasing pH. The inhibition of the cofilin binding rate increases with the time of BeFx incubation with F-actin, indicating the existence of two BeFx-F-actin complexes. Cofilin dissociates BeFx from the filament, while BeFx does not bind to F-actin saturated with cofilin, presumably because of the cofilin-induced changes in the nucleotide-binding cleft of F-actin. These changes are apparent from the increase in the fluorescence intensity of F-actin bound -ADP upon cofilin binding and a decrease in its accessibility to collisional quenchers. BeFx also affects the nucleotide-binding cleft of F-actin, as indicated by an increase in the fluorescence intensity of -ADP-F-actin. Phalloidin and cofilin inhibit, but do not exclude each other binding to their complexes with F-actin. Phalloidin promotes the dissociation of cofilin from F-actin and slowly reverses the cofilin-induced disorder in the DNase I binding loop of subdomain 2.