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Phosphorylation of the cardiac troponin complex by PKA at S22 and S23 of troponin I (TnI) accelerates Ca(2+) release from troponin C (TnC). The region of TnI around the bisphosphorylation site binds to, and stabilizes, the Ca(2+) bound N-terminal domain of TnC. Phosphorylation interferes with this interaction between TnI and TnC resulting in weaker Ca(2+) binding. In this study, we used (1)H-(15)N-HSQC NMR to investigate at the atomic level the interaction between an N-terminal fragment of TnI consisting of residues 1-64 of TnI (I1-64) and TnC. We produced several mutants of I1-64, TnI, and TnC to test the contribution of certain residues to the transmission of the phosphorylation signal in both NMR experiments and functional assays. We also investigated how phosphorylation of the PKC sites in I1-64 (S41 and S43) affects the interaction of I1-64 with TnC. We found that phosphorylation of S22 and S23 produced only localized effects in the structure of I1-64 between residues 24 and 34. Residues 1-17 of I1-64 did not bind to TnC, and residues 38-64 bound tightly to the C-terminal domain of TnC regardless of phosphorylation. Residues 22-34 bound weakly to TnC in a phosphorylation sensitive manner. Bisphosphorylation prevented this phosphorylation switch region from interacting with TnC. Systematic mutation of residues in the phosphorylation switch did not prevent PKA phosphorylation from accelerating Ca(2+) release from troponin. We conclude that the phosphorylation switch binds to TnC via an extended interaction site spanning residues R19 to A34.

The N-terminal extension of cardiac troponin I (TnI) is bisphosphorylated by protein kinase A in response to beta-adrenergic stimulation. How this signal is transmitted between TnI and troponin C (TnC), resulting in accelerated Ca(2+) release, remains unclear. We recently proposed that the unphosphorylated extension interacts with the N-terminal domain of TnC stabilizing Ca(2+) binding and that phosphorylation prevents this interaction. We now use (1)H NMR to study the interactions between several N-terminal fragments of TnI, residues 1-18 (I1-18), residues 1-29 (I1-29), and residues 1-64 (I1-64), and TnC. The shorter fragments provide unambiguous information on the N-terminal regions of TnI that interact with TnC: I1-18 does not bind to TnC whereas the C-terminal region of unphosphorylated I1-29 does bind. Bisphosphorylation greatly weakens this interaction. I1-64 contains the phosphorylatable N-terminal extension and a region that anchors I1-64 to the C-terminal domain of TnC. I1-64 binding to TnC influences NMR signals arising from both domains of TnC, providing evidence that the N-terminal extension of TnI interacts with the N-terminal domain of TnC. TnC binding to I1-64 broadens NMR signals from the side chains of residues immediately C-terminal to the phosphorylation sites. Binding of TnC to bisphosphorylated I1-64 does not broaden these NMR signals to the same extent. Circular dichroism spectra of I1-64 indicate that bisphosphorylation does not produce major secondary structure changes in I1-64. We conclude that bisphosphorylation of cardiac TnI elicits its effects by weakening the interaction between the region of TnI immediately C-terminal to the phosphorylation sites and TnC either directly, due to electrostatic repulsion, or via localized conformational changes.

Phosphorylation of the unique N-terminal extension of cardiac troponin I (TnI) by PKA modulates Ca(2+) release from the troponin complex. The mechanism by which phosphorylation affects Ca(2+) binding, however, remains unresolved. To investigate this question, we have studied the interaction of a fragment of TnI consisting of residues 1-64 (I1-64) with troponin C (TnC) by isothermal titration microcalorimetry and cross-linking. I1-64 binds extremely tightly to the C-terminal domain of TnC and weakly to the N-terminal domain. Binding to the N-domain is weakened further by phosphorylation. Using the heterobifunctional cross-linker benzophenone-4-maleimide and four separate cysteine mutants of I1-64 (S5C, E10C, I18C, R26C), we have probed the protein-protein interactions of the N-terminal extension. All four I1-64 mutants cross-link to the N-terminal domain of TnC. The cross-linking is enhanced by Ca(2+) and reduced by phosphorylation. By introducing the same monocysteine mutations into full-length TnI, we were able to probe the environment of the N-terminal extension in intact troponin. We find that the full length of the extension lies in close proximity to both TnC and troponin T (TnT). Ca(2+) enhances the cross-linking to TnC. Cross-linking to both TnC and TnT is reduced by prior phosphorylation of the TnI. In binary complexes the mutant TnIs cross-link to both the isolated TnC N-domain and whole TnC. Cyanogen bromide digestion of the covalent TnI-TnC complex formed from intact troponin demonstrates that cross-linking is predominantly to the N-terminal domain of TnC.