ACTINS VIEW OF ACTOMYOSIN INTERFACE

Actomyosin interactions were examined by using yeast actin mutants wit h alanines replacing charged amino acid pairs D24/D25, E99/E100, D80/D 81, and E83/K84. In the in vitro motility experiments, actin filaments of D24A/D25A or E99A/E100A mutants moved in the presence of 0.7% meth ylcellulose at the velocities of wild-type actin. Without methylcellul ose, these mutant filaments, but not the D80/D81 or E83/K84 filaments, dissociated from the assay surface upon addition of ATP. Measurements of myosin subfragment-1 (S1) binding to D24A/D25A- and E99A/E100A-pol ymerized actins in the presence of ATP revealed a three- and twofold d ecrease in their binding constant, respectively, compared with wild-ty pe actin. In contrast to this, all monomeric actins had the same bindi ng affinity for S1. The rates and extents of polymerization of D24A/D2 5A and E99A/E100A actins by S1 were reduced in comparison to wild-type actin. The local structure of subdomain-e on actin, as probed by subt ilisin cleavage, was not altered for either mutant. A twofold decrease in nucleotide exchange was detected for the D24A/D25A mutant actin. T hese results demonstrate the involvement of the D24/D25 and E99/E100 r esidues in the weak binding of myosin to actin and reveal that residue s D80/D81 and E83/K84 do not modulate actomyosin interactions.