Mapping the binding site of thymosin beta(4) on actin by competition with G-actin binding proteins indicates negative co-operativity between binding sites located on opposite subdomains of actin

The beta-thymosins are small monomeric (G-)actin-binding proteins of 5 kDa that are supposed to act intracellularly as actin-sequestering factors stabilizing the cytoplasmic monomeric pool of actin. The binding region of thymosin beta(4) was determined by analysing the binding of thymosin beta(4) to actin complexed with DNase I, gelsolin or gelsolin segment 1. Binding was analysed by determining the increase in the critical concentration of actin polymerization by native gel electrophoresis or chemical cross-linking. The formation of a ternary complex including thymosin beta(4) should indicate that the actin-binding proteins attach to different sites on actin. Competition would be indicative of binding to identical or overlapping sites on actin or of a negative co-operative linkage between the two binding sites. Competition of thymosin beta(4) for actin binding was observed in the presence of intact gelsolin or the N-terminal gelsolin fragment, segment 1, indicating that thymosin beta(4) binds to a site close to or identical with the gelsolin segment 1-binding-site. The ternary complex of actin-DNase I-thymosin beta(4) was obtained only when using the chemically cross-linked actin-thymosin beta(4) complex, indicating that thymosin beta(4) is dissociated by the binding of DNase I to actin. It is suggested that the dissociation of thymosin beta(4) by DNase I binding to actin is caused by negative co-operativity between their spatially separated binding sites on actin. A similar negative co-operativity was observed between DNase I and gelsolin segment 1 binding to actin. The results therefore indicate that the respective binding sites for DNase I and segment 1 on subdomains 1 and 2 of actin are linked in a negative co-operative manner.