J. Moraczewska1, B. Wawro1, S.Yu. Khaitlina2, A. Wozniak1 and H. Strzelecka-Golaszewska2
1Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland, and 2Institute of Cytology, 194064 St Petersburg, Russia
Current F-actin models suggest a shift of subdomains 2 and 4 closer together, whereby the cleft between them narrows, and a rearrangement of the DNase- I-binding loop on the top of subdomain 2 relative to the crystal G-actin structure. We have used limited proteolysis method and fluorescent labels dansyl cadaverine or dansyl ethylenediamine covalently attached to Gln41 to compare changes in the structure of subdomain 2 of actin accompanying polymerization with those induced in G-actin by addition of polymerizing salts and by various treatments influencing the kinetics of polymerization. These latter included exchange of the tightly bound Ca2+ for Mg2+, substitution of ADP for ATP in Mg-G-actin, and treatment with phalloidin which, as we show here, not only accelerates salt-induced polymerization but also promotes polymerization of actin in low ionic strength solutions. The results suggest that the closure of the interdomain cleft precedes polymerization. The subdomain shifts underlying this change seem to be a prerequisite of the polymer formation, and this F-like conformation is then stabilized by intermonomer interactions. Consistent with evidence from electron microscopic and biochemical studies in other laboratories, our results suggest that the rearrangement of the DNase-I-binding loop is a result of ATP hydrolysis and release of phosphate following the polymer formation.
In the light of our data, the subdomain shifts promoted by the binding of Mg2+ in the interdomain cleft as well as changes observed on phalloidin binding to G-actin can be regarded as a part of the monomer activation, the first step in the polymerization process.