Vl. Korman et al., An actin subdomain 2 mutation that impairs thin filament regulation by troponin and tropomyosin, J BIOL CHEM, 275(29), 2000, pp. 22470-22478
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.