Calcium plays distinctive structural roles in the N- and C-terminal domains of cardiac troponin C.
One- and two-dimensional NMR techniques were used to compare the structural consequences of Ca2+ binding to both the low and high affinity Ca2+ binding sites in recombinant cardiac troponin C (cTnC3). In the absence of Ca2+, the short beta-sheet located between the high affinity Ca2+/Mg2+ binding sites in the C-terminal domain was found to be absent or loosely formed as judged by the inter-residue NOEs and chemical shifts of resonances in the Ca2+ binding loops. In contrast, the N-terminal domain beta-sheet located between site II and the naturally inactive site I was present even in the absence of bound Ca2+. Calcium-binding mutant proteins having either an inactive Ca2+ binding site III (CBM-III) or an inactive Ca2+ binding site IV (CBM-IV) (Negele, J. C., Dotson, D., Liu, W., Sweeney, H. L., and Putkey, J. A. (1992) J. Biol. Chem. 267, 825-831) were used to study the structural consequences of Ca2+ binding to each of the high affinity sites located in the C-terminal domain. Only a single active Ca2+ binding site was found necessary for formation of the short beta-sheet between Ca2+ binding sites III and IV. However, the absence of bound Ca2+ at site III was found to produce greater instability in the C-terminal domain as judged from the mobility of the C-terminal aromatic hydrophobic cluster. Thus, Ca2+ binding to the high affinity sites in the C-terminal domain results in an ordering of the aromatic hydrophobic cluster, as well as formation of a short beta-sheet between Ca2+ binding sites III and IV. These results demonstrate that Ca2+ binding plays distinctive structural roles in the N- and C-terminal domains of cTnC.
Brito
R M
RM
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
Krudy
G A
GA
Negele
J C
JC
Putkey
J A
JA
Rosevear
P R
PR
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000378:metabolism; D009206:Myocardium / Q000737:chemistry*; D011487:Protein Conformation; D014336:Troponin / Q000737:chemistry* / Q000378:metabolism; D019209:Troponin C
D008670:Metals; D014336:Troponin; D019209:Troponin C; D002118:Calcium