An actin subdomain 2 mutation that impairs thin filament regulation by troponin and tropomyosin.
Striated muscle thin filaments adopt different quaternary structures, depending upon calcium binding to troponin and myosin binding to actin. Modification 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 issue. The mutation increased the affinity of tropomyosin for actin 3-fold. The addition of Ca(2+) to mutant actin filaments containing troponin-tropomyosin produced little increase in the thin filament-myosin S1 MgATPase rate. Despite this, three-dimensional reconstruction of electron microscope images of filaments in the presence of troponin and Ca(2+) 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 comparable affinity to mutant and wild type actin in the absence and presence of 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 of 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 changes in the actin-troponin-tropomyosin complex.
Korman
V L
VL
Departments of Biochemistry and Internal Medicine, University of Iowa, College of Medicine, Iowa City, Iowa 52242, USA.
Hatch
V
V
Dixon
K Y
KY
Craig
R
R
Lehman
W
W
Tobacman
L S
LS
D008841:Actin Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D000199:Actins / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D009119:Muscle Contraction; D009154:Mutation; D011485:Protein Binding; D017510:Protein Folding; D012441:Saccharomyces cerevisiae; D014335:Tropomyosin / Q000378:metabolism*; D014336:Troponin / Q000378:metabolism*
D000199:Actins; D014335:Tropomyosin; D014336:Troponin