Antagonistic effects of cofilin, beryllium fluoride complex and phalloidin on subdomain 2 and nucleotide-binding cleft in F-actin

¹ Hebrew Univ. Hadassah Sch. Dental Med.
² Hebrew University of Jerusalem
³ UCLA

^* To whom correspondence should be addressed. E-mail: muhlrad{at}cc.huji.ac.il.

Submitted on April 25, 2006
Revised on June 6, 2006
Accepted on 31 August 2006

	Abstract

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.

Key Words: actin, beryllium fluoride complex, cofilin, limited proteolysis, phalloidin