The binding of W7, an inhibitor of striated muscle contraction, to cardiac troponin C.
W7 is a well-characterized calmodulin antagonist. It decreases the maximal tension and rate of ATP hydrolysis in cardiac muscle fibers. Cardiac troponin C (cTnC) has been previously implicated as the mechanistically significant target for W7 in the myofilament. Two-dimensional NMR spectra ({1H,15N}- and {1H,13C}-HSQCs) were used to monitor the Ca2+-dependent binding of W7 to cTnC. Titration of cTnC x 3Ca2+ with W7 indicated binding to both domains of the protein. We examined the binding of W7 to the separated domains of cTnC to simplify the spectral analysis. In the titration of the C-terminal domain (cCTnC x 2Ca2+), the spectral peaks originating from a subset of residues changed nonuniformly, and could not be well-described as single-site binding. A global fit of the cCTnC x 2Ca2+ titration data to a two-site, sequential binding model (47 residues simultaneously fit) yielded a dissociation constant (Kd1) of 0.85-0.91 mM for the singly bound state, with the second dissociation constant fit to 3.40-3.65 mM (> or = 4 x Kd1). The titration data for the N-terminal domain (cNTnC x Ca2+) was globally fit to single-site binding model with a Kd of 0.15-0.30 mM (41 residues fit). The data are consistent with W7 binding to each domain's major hydrophobic pocket, coordinating side chains responsible for liganding cTnI. When in muscle fibers, W7 may compete with cTnI for target sites on cTnC.
Hoffman
Ryan M B
RM
CIHR Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Li
Monica X
MX
Sykes
Brian D
BD
D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009200:Myocardial Contraction / Q000187:drug effects*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D013449:Sulfonamides / Q000378:metabolism* / Q000494:pharmacology; D019209:Troponin C / Q000378:metabolism*
D013449:Sulfonamides; D019209:Troponin C; C017967:W 7