Concerted action of the high affinity calcium binding sites in skeletal muscle troponin C.
Mutants of each of the four divalent cation binding sites of chicken skeletal muscle troponin C (TnC) were constructed using site-directed mutagenesis to convert Asp to Ala at the first coordinating position in each site. With a view to evaluating the importance of site-site interactions both within and between the N- and C-terminal domains, in this study the mutants are examined for their ability to associate with other components of the troponin-tropomyosin regulatory complex and to regulate thin filaments. The functional effects of each mutation in reconstitution assays are largely confined to the domain in which it occurs, where the unmutated site is unable to compensate for the defect. Thus the mutants of sites I and II bind to the regulatory complex but are impaired in ability to regulate tension and actomyosin ATPase activity, whereas the mutants of sites III and IV regulate activity but are unable to remain bound to thin filaments unless Ca2+ is present. When all four sites are intact, free Mg2+ causes a 50-60-fold increase in TnC's affinity for the other components of the regulatory complex, allowing it to attach firmly to thin filaments. Calcium can replace Mg2+ at a concentration ratio of 1:5000, and at this ratio the Ca2.TnC complex is more tightly bound to the filaments than the Mg2.TnC form. In the C-terminal mutants, higher concentrations of Ca2+ (above tension threshold) are required to effect this transformation than in the recombinant wild-type protein, suggesting that the mutants reveal an attachment mediated by Ca2+ in the N-domain sites.
Sorenson
M M
MM
Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Brazil.
da Silva
A C
AC
Gouveia
C S
CS
Sousa
V P
VP
Oshima
W
W
Ferro
J A
JA
Reinach
F C
FC
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D008274:Magnesium / Q000378:metabolism; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D014336:Troponin / Q000235:genetics / Q000378:metabolism*; D019209:Troponin C
D014336:Troponin; D019209:Troponin C; D008274:Magnesium; D002118:Calcium