Mutations in actin subdomain 3 that impair thin filament regulation by troponin and tropomyosin.
Thin filament-mediated regulation of striated muscle contraction involves conformational switching among a few quaternary structures, with transitions induced by binding of Ca(2+) and myosin. We establish and exploit Saccharomyces cerevisiae actin as a model system to investigate this process. Ca(2+)-sensitive troponin-tropomyosin binding affinities for wild type yeast actin are seen to closely resemble those for muscle actin, and these hybrid thin filaments produce Ca(2+)-sensitive regulation of the myosin S-1 MgATPase rate. Yeast actin filament inner domain mutant K315A/E316A depresses Ca(2+) activation of the MgATPase rate, producing a 4-fold weakening of the apparent Ca(2+) affinity and a 50% decrease in the MgATPase rate at saturating Ca(2+) concentration. Observed destabilization of troponin-tropomyosin binding to actin in the presence of Ca(2+), a 1.4-fold effect, provides a partial explanation. Despite the decrease in apparent MgATPase Ca(2+) affinity, there was no detectable change in the true Ca(2+) affinity of the thin filament, measured using fluorophore-labeled troponin. Another inner domain mutant, E311A/R312A, decreased the MgATPase rate but did not change the apparent Ca(2+) affinity. These results suggest that charged residues on the surface of the actin inner domain are important in Ca(2+)- and myosin-induced thin filament activation.
Korman
V L
VL
Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242, USA.
Tobacman
L S
LS
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000409:Alanine / Q000235:genetics; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D008956:Models, Chemical; D009119:Muscle Contraction / Q000502:physiology; D009154:Mutation; D015879:Myosin Subfragments / Q000378:metabolism; D009994:Osmolar Concentration; D011485:Protein Binding; D017186:Titrimetry; D014335:Tropomyosin / Q000378:metabolism*; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C / Q000378:metabolism
D000199:Actins; D015879:Myosin Subfragments; D014335:Tropomyosin; D014336:Troponin; D019209:Troponin C; D017301:Ca(2+) Mg(2+)-ATPase; D000409:Alanine; D002118:Calcium