An actin subdomain 2 mutation that impairs thin filament regulation by troponin and tropomyosin

Striated muscle thin filaments adopt different quaternary structures, depen ding upon calcium binding to troponin and myosin binding to actin. Modifica tion of actin subdomain 2 alters troponin-tropomyosin-mediated regulation, suggesting that this region of actin may contain important protein-protein interaction sites. We used yeast actin mutant D56A/E57A to examine this iss ue. The mutation increased the affinity of tropomyosin for actin 3-fold. Th e addition of Ca2+ to mutant actin filaments containing troponin-tropomyosi n produced little increase in the thin filament-myosin S1 MgATPase rate. De spite this, three-dimensional reconstruction of electron microscope images of filaments in the presence of troponin and Ca2+ showed tropomyosin to be in a position similar to that found for muscle actin filaments, where most of the myosin binding site is exposed. Troponin-tropomyosin bound with comp arable affinity to mutant and wild type actin in the absence and presence o f calcium, and in the presence of myosin S1, tropomyosin bound very tightly to both types of actin. The mutation decreased actin-myosin S1 affinity 13 -fold in the presence of troponin-tropomyosin and 2.6-fold in the absence o f the regulatory proteins. The results suggest the importance of negatively charged actin subdomain 2 residues 56 and 57 for myosin binding to actin, for tropomyosin-actin interactions, and for regulatory conformational chang es in the actin-troponin-tropomyosin complex.