Tropomyosin-Binding Site(s) on the Dictyostelium Actin Surface As Identified by Site-Directed Mutagenesis

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Abstract
To identify tropomyosin-binding site(s) on the surface of actin molecule, we examined the effect of mutagenesis introduced to subdomain 4 of actin. Because the sequence of Gln228−Ser232 of Dictyostelium actin differs from that of Tetrahymena actin that does not bind tropomyosin, the Dictyostelium/Tetrahymena chimeric actin was produced. Also, Lys238 and Glu241 were replaced with alanine (mutant 645) to study the role of charged residues which are located at both ends of a β-sheet. As a control experiment, a negative charge was introduced near to the N-terminus (mutant 663). To facilitate the separation of mutant actins without affecting the normal function, Glu360 was replaced with histidine. As a control mutant to such mutants, the mutant 647 (E360H) was produced. Mutant actins were expressed in Dictyostelium cells. All mutant actins were functional: they (i) polymerize and (ii) activate ATPase activity of rabbit skeletal myosin subfragment-1 (S1). The mutant 663 (G2E) showed tropomyosin binding and activated myosin ATPase almost as well as rabbit skeletal actin. However, the tropomyosin binding of the mutant 645 (K238A/E241A/E360H) became magnesium dependent. The chimeric actin (mutant 646: QTAAS-to-KAYKE replacement and E360H) showed decreased tropomyosin binding even in the presence of magnesium ions. These results indicate that the tropomyosin-binding sites of “on”-state actin are on subdomain 4. Surprisingly, the chimeric actin showed more cooperative calcium regulation than rabbit skeletal actin in the presence of tropomyosin−troponin. The mutant actin 645 can hardly activate S1 ATPase irrespective of calcium concentration in the presence of tropomyosin−troponin, even though this actin by itself can activate S1 ATPase. The steric blocking or cooperative/allosteric mechanism of thin filament regulation is discussed.