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

Citation
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
Citations number
53
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
00219258 → ACNP
Volume
275
Issue
29
Year of publication
2000
Pages
22470 - 22478
Database
ISI
SICI code
0021-9258(20000721)275:29<22470:AAS2MT>2.0.ZU;2-F
Abstract
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.