Conformational exchange has been demonstrated within the regulatory domain of calcium-saturated cardiac troponin C when bound to the NH 2 -terminal domain of cardiac troponin I-(1-80), and cardiac troponin I-(1-80)DD, having serine residues 23 and 24 mutated to aspartate to mimic the phosphorylated form of the protein. Binding of cardiac troponin I-(1-80) decreases conformational exchange for residues 29, 32, and 34. Comparison of average transverse cross correlation rates show that both the NH 2 -and COOH-terminal domains of cardiac troponin C tumble with similar correlation times when bound to cardiac troponin I-(1-80). In contrast, the NH 2 -and COOH-terminal domains in free cardiac troponin C and cardiac troponin C bound cardiac troponin I-(1-80)DD tumble independently. These results suggest that the nonphosphorylated cardiac specific NH 2 terminus of cardiac troponin I interacts with the NH 2 -terminal domain of cardiac troponin C. Conformational changes induced by Ca 2ϩ binding to the NH 2 -terminal domain of skeletal TnC (sTnC) have been followed by both x-ray (6) and solution NMR (7). For the free skeletal TnC protein, Ca 2ϩ binding results in a conformational change from the "closed" form to an "open" form exposing a patch of hydrophobic residues for interaction with TnI (7). Surprisingly, the Ca 2ϩ -bound NH 2 -terminal domain of cTnC was found to maintain a closed conformation (8). The solution structure of the Ca 2ϩ saturated regulatory domain of human cTnC, cTnC-(1-91), in 9% trifluoroethanol, also reveals a "closed" conformation with little exposed hydrophobic surface (9). In addition, 15 To elucidate the role of phosphorylation of the cardiac-specific amino terminus of cTnI on full-length cTnC and to unequivocally demonstrate the presence of conformational exchange in the regulatory domain of full-length cTnC, we have probed the dynamics of cTnC free and bound to cTnI-(1-80) and cTnI-(1-80)DD. We show that residues within inactive Ca 2ϩ binding site I undergo chemical exchange consistent with an equilibrium between closed and opened forms both in the presence and absence of the NH 2 -terminal cTnI domain. Based on chemical shift perturbation mapping and transverse relaxation rates, the cardiac-specific NH 2 terminus of cTnI-(1-80) appears to make additional interactions with the regulatory domain of cTnC, which results in slowing down the exchange rate in the defunct Ca 2ϩ binding site I as evidenced by the presence of . These interactions were not observed in the free protein or in the complex with cTnI-(1-80)DD. MATERIALS AND METHODSProteins-The single cysteine form of cTnC (cTnC35S) was used in the study (10, 11). 15 N and 2 H labeling was accomplished using minimal medium containing 90% 2 H 2 O and 1 g/liter 15 NH 4 Cl. Cardiac troponin C was purified as described previously (5). Cardiac troponin I-(1-80) and cTnI-(1-80)DD were expressed as inclusion bodies purified in 8 M urea.2 All proteins were judged to be homogeneous by SDS-polyacrylamide gel electrophoresis and staining with Coomassie...

Phosphorylation of the cardiac specific amino-terminus of troponin I has been demonstrated to reduce the Ca 2+

Adrenergic stimulation induces positive changes in cardiac contractility and relaxation. Cardiac troponin I is phosphorylated at different sites by protein kinase A and protein kinase C, but the effects of these post-translational modifications on the rate and extent of contractility and relaxation during ␤-adrenergic stimulation in the intact animal remain obscure. To investigate the effect(s) of complete and chronic cTnI phosphorylation on cardiac function, we generated transgenic animals in which the five possible phosphorylation sites were replaced with aspartic acid, mimicking a constant state of complete phosphorylation (cTnI-AllP). We hypothesized that chronic and complete phosphorylation of cTnI might result in increased morbidity or mortality, but complete replacement with the transgenic protein was benign with no detectable pathology. To differentiate the effects of the different phosphorylation sites, we generated another mouse model, cTnI-PP, in which only the protein kinase A phosphorylation sites (Ser 23 /Ser 24 ) were mutated to aspartic acid. In contrast to the cTnIAllP, the cTnI-PP mice showed enhanced diastolic function under basal conditions. The cTnI-PP animals also showed augmented relaxation and contraction at higher heart rates compared with the nontransgenic controls. Nuclear magnetic resonance amide proton/nitrogen chemical shift analysis of cardiac troponin C showed that, in the presence of cTnI-AllP and cTnI-PP, the N terminus exhibits a more closed conformation, respectively. The data show that protein kinase C phosphorylation of cTnI plays a dominant role in depressing contractility and exerts an antithetic role on the ability of protein kinase A to increase relaxation.Recent studies have demonstrated that changes in the phosphorylation states of key cardiac regulatory proteins can have dramatic effects on normal cardiac function (1, 2). Phosphorylation of cardiac troponin I (cTnI), 1 a thin filament regulatory protein, may be particularly important in modulating cardiac function. cTnI, together with cardiac troponin T (cTnT) and cardiac troponin C (cTnC), form the troponin complex. The protein binds to both cTnC and actin and is a critical component in activating contraction as it serves as the Ca 2ϩ -sensing apparatus. Within the cardiac isoform's amino-terminal extension, serines are present at residues 23 and 24 (Ser 23 /Ser 24 ), which serve as substrates for protein kinase A (PKA), which is activated in response to ␤-adrenergic stimulation of the heart (3). Several investigations report that PKA-mediated phosphorylation of cTnI results in a reduction in myofilament Ca 2ϩ sensitivity (4), an increase in cross-bridge cycling (5), and increased binding of cTnI to the thin filament. Cardiac TnI is also a substrate for protein kinase C (PKC) phosphorylation at Ser 43 /Ser 45 and Thr 144 (position 143 in the human protein) (6). However, the substrate specificity of these sites is not absolute, since PKC can phosphorylate the PKA sites (7-9). Whereas PKA-mediated phosphorylation is th...

The N-terminal domain of cardiac troponin I (cTnI) comprising residues 33-80 and lacking the cardiac-specific amino terminus forms a stable binary complex with the C-terminal domain of cardiac troponin C (cTnC) comprising residues 81-161. We have utilized heteronuclear multidimensional NMR to assign the backbone and side-chain resonances of Ca2+-saturated cTnC(81-161) both free and bound to cTnI(33-80). No significant differences were observed between secondary structural elements determined for free and cTnI(33-80)-bound cTnC(81-161). We have determined solution structures of Ca2+-saturated cTnC(81-161) free and bound to cTnI(33-80). While the tertiary structure of cTnC(81-161) is qualitatively similar to that observed free in solution, the binding of cTnI(33-80) results mainly in an opening of the structure and movement of the loop region between helices F and G. Together, these movements provide the binding site for the N-terminal domain of cTnI. The putative binding site for cTnI(33-80) was determined by mapping amide proton and nitrogen chemical shift changes, induced by the binding of cTnI(33-80), onto the C-terminal cTnC structure. The binding interface for cTnI(33-80), as suggested from chemical shift changes, involves predominantly hydrophobic interactions located in the expanded hydrophobic pocket. The largest chemical shift changes were observed in the loop region connecting helices F and G. Inspection of available TnC sequences reveals that these residues are highly conserved, suggesting a common binding motif for the Ca2+/Mg2+-dependent interaction site in the TnC/TnI complex.