Interaction of cardiac troponin C with calmodulin antagonist [corrected] W7 in the presence of three functional regions of cardiac troponin I.
W7 is a well-known calmodulin (CaM) antagonist and has been implicated as an inhibitor of the troponin C-mediated Ca(2+) activation of cardiac muscle contraction. In this study, we use NMR spectroscopy to study binding of W7 to cardiac troponin C (cTnC) free or in complex with cardiac troponin I (cTnI) peptides. Titration of cTnC.3Ca(2+) with W7 shows that residues throughout the sequence, including the N- and C-domains of cTnC and the central linker, are affected. Analysis of the binding stoichiometry and the trajectories of chemical shift changes indicate that W7 binding occurs at multiple sites. To address the issue of whether multiple-site binding is relevant within the troponin complex, W7 is titrated to a cTnC-cTnI complex (cTnC.3Ca(2+).cTnI(34)(-)(71).cTnI(128)(-)(163)). In the presence of the N-terminal (residues approximately 34-71), inhibitory (residues approximately 128-147), and switch (residues approximately 147-163) regions of cTnI, W7 induces chemical shift changes only in the N-domain and not in the C-domain or the central linker of cTnC. The results indicate that in the presence of cTnI, W7 no longer binds to multiple sites of cTnC but instead binds specifically to the N-domain, and the binding (K(D) = 0.5 +/- 0.1 mM) can occur together with the switch region of cTnI. Hence, W7 may play a role in directly modulating the Ca(2+) sensitivity of the regulatory domain of cTnC and the interaction of the switch region of cTnI and cTnC.
Li
Monica X
MX
CIHR Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Hoffman
Ryan M B
RM
Sykes
Brian D
BD
D002118:Calcium / Q000378:metabolism; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors; D006801:Humans; D009206:Myocardium / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013449:Sulfonamides / Q000378:metabolism*; D017186:Titrimetry; D019209:Troponin C / Q000378:metabolism*; D019210:Troponin I / Q000737:chemistry* / Q000378:metabolism*
D004791:Enzyme Inhibitors; D010455:Peptides; D013449:Sulfonamides; D019209:Troponin C; D019210:Troponin I; C017967:W 7; D002118:Calcium