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Role of charged amino acid pairs in subdomain-1 of actin in interactions with myosin.

Yeast actin mutants with alanines replacing charged amino acid pairs D24/D25, E99/E100, D80/D81, and E83/K84 were studied to assess their role in interactions with myosin. In a previous report Dictyostelium actin filaments with residues D24/D25 or E99/E100 replaced with histidines showed complete or partial loss of filament sliding in the in vitro motility assay [Johara, M., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2127-2131]. In the motility experiments reported here, actin filaments with alanines substituted at D24/D25 or E99/E100 moved in the presence of 0.7% methylcellulose at velocities similar to those of wild-type yeast actin. Without methylcellulose, mutant filaments dissociated from the assay surface upon addition of ATP with little or no sliding detected. In contrast to this, filaments with alanines substituted at D80/D81 or E83/K84 were motile in the presence and absence of methylcellulose. Direct binding measurements involving cosedimentation of D24A/D25A and E99A/E100A actins with myosin subfragment-1 (S-1) in the presence of ATP revealed 3- and 2-fold decreases in their binding constants, respectively, compared to wild-type actin. In the absence of ATP all yeast actins had a similar affinity for S-1. A large decrease in the activation of S-1 ATPase was observed for both D24A/D25A and E99A/E100A actins. The D80A/D81A and E83A/K84A actin filaments showed normal S-1 binding and activation of ATPase activity. These results demonstrate the involvement of the D24/D25 and E99/E100 charged residues in the weak binding of myosin to actin and reveal that D80/D81 and E83/K84 residues in the 79-92 helix do not modulate actomyosin interactions.