Actin-actin contact: inhibition of actin-polymerization by subdomain 4 peptide fragments

F-Actin was digested with alpha-chymotrypsin in 6 M urea, and two peptide fragments from subdomain 4 of actin molecule [Kabsch, W., Mannherz, H.G., Suck, D., Pai, E.F., & Holmes K.C. (1990) Nature 347, 37-44] were purified by reverse-phase HPLC and Sephadex G-50 gel filtration. The peptide fragments were identified as segments from Arg-177 to Tyr-198 (2.6-kDa peptide) and from Ser-199 to Tyr-279 (9.1-kDa peptide). Their effects on actin polymerization induced by 50 or 100 mM KCl were studied by measuring the increase in viscosity by the falling ball method. The 2.6-kDa peptide decreased the rate of actin polymerization and increased the critical concentration for the polymerization. Based on the atomic model of the actin filament [Holmes, K.C., Popp, D., Gebhard, W., & Kabsch, W. (1990) Nature 347, 44-49], the peptide is presumed to bind to the barbed end of the actin filament and inhibit the polymerization. By assuming that the peptide affected the rate of association of the actin monomer to the end of the actin filament, well-fitting curves for the polymerization kinetics were calculated. Computer-assisted results indicated that the dissociation constant of the 2.6-kDa peptide for F-actin is 200 to 260 microM. In contrast, the 9.1-kDa peptide only slightly inhibited actin polymerization. These results suggest that the actin-actin interface in the region between Arg-177 and Tyr-198 has a stronger interaction than those between Ser-199 and Tyr-279. The amino acid sequence L-T-D-Y-L present in the 2.6-kDa segment is homologous to a common sequence in the F-actin capping domain of various actin-binding proteins.