Characterization of the interaction between the N-terminal extension of human cardiac troponin I and troponin C

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 Ca2+ release, remains unclear. We recently proposed that the unphosphorylated extension interacts with the N-terminal domain of TnC stabilizing Ca2+ binding and that phosphorylation prevents this interaction. We now use H-1 NMR to study the interactions between several N-terminal fragments of TnI, residues 1 - 18 (11 - 18), residues 1 - 29 (11 - 29), and residues 1 - 64 (11 - 64), and TnC. The shorter fragments provide unambiguous information on the N-terminal regions of TnI that interact with TnC: 11 - 18 does not bind to TnC whereas the C-terminal region of unphosphorylated 11 - 29 does bind. Bisphosphorylation greatly weakens this interaction. 11 - 64 contains the phosphorylatable N-terminal extension and a region that anchors 11 - 64 to the C-terminal domain of TnC. 11 - 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 11 - 64 broadens NMR signals from the side chains of residues immediately C-terminal to the phosphorylation sites. Binding of TnC to bisphosphorylated 11 - 64 does not broaden these NMR signals to the same extent. Circular dichroism spectra of 11 - 64 indicate that bisphosphorylation does not produce major secondary structure changes in 11 - 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.