The cardiac-specific N-terminal region of troponin I positions the regulatory domain of troponin C.

The cardiac isoform of troponin I (cTnI) has a unique 31-residue N-terminal region that binds cardiac troponin C (cTnC) to increase the calcium sensitivity of the sarcomere. The interaction can be abolished by cTnI phosphorylation at Ser22 and Ser23, an important mechanism for regulating cardiac contractility. cTnC contains two EF-hand domains (the N and C domain of cTnC, cNTnC and cCTnC) connected by a flexible linker. Calcium binding to either domain favors an "open" conformation, exposing a large hydrophobic surface that is stabilized by target binding, cTnI[148-158] for cNTnC and cTnI[39-60] for cCTnC. We used multinuclear multidimensional solution NMR spectroscopy to study cTnI[1-73] in complex with cTnC. cTnI[39-60] binds to the hydrophobic face of cCTnC, stabilizing an alpha helix in cTnI[41-67] and a type VIII turn in cTnI[38-41]. In contrast, cTnI[1-37] remains disordered, although cTnI[19-37] is electrostatically tethered to the negatively charged surface of cNTnC (opposite its hydrophobic surface). The interaction does not directly affect the calcium binding affinity of cNTnC. However, it does fix the positioning of cNTnC relative to the rest of the troponin complex, similar to what was previously observed in an X-ray structure [Takeda S, et al. (2003) Nature 424(6944):35-41]. Domain positioning impacts the effective concentration of cTnI[148-158] presented to cNTnC, and this is how cTnI[19-37] indirectly modulates the calcium affinity of cNTnC within the context of the cardiac thin filament. Phosphorylation of cTnI at Ser22/23 disrupts domain positioning, explaining how it impacts many other cardiac regulatory mechanisms, like the Frank-Starling law of the heart.

The role of electrostatics in the interaction of the inhibitory region of troponin I with troponin C.

We have addressed the electrostatic interactions occurring between the inhibitory region of cardiac troponin I with the C-lobe of troponin C using scanning glycine mutagenesis of the inhibitory region. We report variations in the electric potentials due to mutation of charged residues within this complex based upon the solved NMR structure (1OZS). These results demonstrate the importance of electrostatics within this complex, and it is proposed that electrostatic interactions are integral to the formation and function of larger ternary troponin complexes. To address this hypothesis, we report (15)N NMR relaxation measurements, which suggest that, within a ternary complex involving the C-lobe and the N-terminal region of troponin I (residues 34-71), the inhibitory region maintains the electrostatic interactions with the E-helix of the C-lobe as observed within the binary complex. These results imply that, in solution, the cardiac troponin complex behaves in a manner consistent with that of the crystal structure of the skeletal isoform (1YTZ). A cardiac troponin complex possessing domain orientations similar to that of the skeletal isoform provides structural insights into altered troponin I activities as observed for the familial hypertrophic cardiomyopathy mutation R144G and phosphorylation of Thr142.