Actin's view of actomyosin interface.
Actomyosin interactions were examined by using yeast actin mutants with alanines replacing charged amino acid pairs D24/D25, E99/E100, D80/D81, 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% methylcellulose at the velocities of wild-type actin. Without methylcellulose, 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-polymerized actins in the presence of ATP revealed a three- and twofold decrease in their binding constant, respectively, compared with wild-type actin. In contrast to this, all monomeric actins had the same binding affinity for S1. The rates and extents of polymerization of D24A/D25A and E99A/E100A actins by S1 were reduced in comparison to wild-type actin. The local structure of subdomain-2 on actin, as probed by subtilisin cleavage, was not altered for either mutant. A twofold decrease in nucleotide exchange was detected for the D24A/D25A mutant actin. These results demonstrate the involvement of the D24/D25 and E99/E100 residues in the weak binding of myosin to actin and reveal that residues D80/D81 and E83/K84 do not modulate actomyosin interactions.
Miller
C J
CJ
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024, USA.
Cheung
P
P
White
P
P
Reisler
E
E
D000199:Actins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D000205:Actomyosin / Q000737:chemistry* / Q000502:physiology; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D066298:In Vitro Techniques; D007700:Kinetics; D015394:Molecular Structure; D009119:Muscle Contraction / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D015879:Myosin Subfragments / Q000737:chemistry; D017354:Point Mutation; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D000199:Actins; D015879:Myosin Subfragments; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000205:Actomyosin