Phosphorylation of Troponin I finely controls the positioning of Troponin for the optimal regulation of cardiac muscle contraction

Troponin is the Ca²⁺ molecular switch that regulates striated muscle contraction. In the heart, troponin Ca²⁺ sensitivity is also modulated by the PKA-dependent phosphorylation of a unique 31-residue N-terminal extension region of the Troponin I subunit (NH₂-TnI). However, the detailed mechanism for the propagation of the phosphorylation signal through Tn, which results in the enhancement of the myocardial relaxation rate, is difficult to examine within whole Tn. Several models exist for how phosphorylation modulates the troponin response in cardiac cells but these are mostly built from peptide-NMR studies and molecular dynamics simulations. Here we used a paramagnetic spin labeling approach to position and track the movement of the NH₂-TnI region within whole Tn. Through paramagnetic relaxation enhancement (PRE)-NMR experiments, we show that the NH₂-TnI region interacts with a broad surface area on the N-domain of the Troponin C subunit. This region includes the Ca²⁺ regulatory Site II and the TnI switch-binding site. Phosphorylation of the NH₂-TnI both weakens and shifts this region to an adjacent site on TnC. Interspin EPR distances between NH₂-TnI and TnC further reveal a phosphorylation induced re-orientation of the TnC N-domain under saturating Ca²⁺ conditions. We propose an allosteric model where phosphorylation triggered cooperative changes in both the interaction of the NH₂-TnI region with TnC, and the re-orientation of the TnC interdomain orientation, together promote the release of the TnI switch-peptide. Enhancement of the myocardial relaxation rate then occurs. Knowledge of this unique role of phosphorylation in whole Tn is important for understanding pathological processes affecting the heart.