The Ca2+ switch of cardiac muscle is modulated by phosphorylation of Ser 22 and 23 in the cardiac specific N-terminus of Troponin I (TnI) by PKA in response to beta-adrenergic stimulation.
It has proposed that the unphosphorylated N terminal peptide of TnI binds to the N terminal lobe of troponin C (TnC) and that the interaction enhances Ca2+ sensitivity. The interaction is abolished by PKA phosphorylation of Ser 22 and 23 reducing Ca2+-sensitivity and increasing the rate of Ca2+ dissociation.
The full structure of cardiac troponin is not known since Takeda's 2003 X-ray structure of the core of cardiac troponin (Nature 424, 35) does not contain several important mobile peptides including the N-terminal 31 amino acids.
Here we show the structural changes of troponin that occur upon phosphorylation using molecular dynamics. We have applied molecular dynamics simulations to an expanded model of the Takeda et al. structure (385 amino acids) that includes the first 31 residues of TnI that we have added in as a linear chain above the TnC N-terminal lobe according to the model of Howarth et al., (J Mol Biol 373, 709).
All simulations have been performed for at least a quarter of a microsecond (with some simulations up to 750 ns) with the AMBER GPU MD package in an isobaric-isothermal, NPT, ensemble.
In the Ca2+-bound unphosphorylated state there is a persistent interaction between Ser 22 and 23 of TnI and Ser 69 and Thr 71 in the Ca2+-binding loop of TnC and the Ca2+ appears strongly coordinated and immobile. The extreme N-terminal 1 to 16 amino acids of TnI are very mobile apart from the two Serines, Lys 20 is the only residue making a persistent interaction with TnC. In the absence of Ca2+ the N-terminal 30 amino acids of cTnI are fully mobile and not interacting with TnC. In addition there are long-range differences in TnI structure due to phosphorylation or removal of Ca2+, centered on the TnI helix II that is partly unwound.
Phosphorylation leads to restructuring after 50 ns with the loss of the TnI Ser 22 and 23 contacts with TnC and a re-orientation of the TnC N-terminal lobe relative to the rest of troponin. Ca2+ becomes more exposed to solvent. Between 100 and 150 ns the Ca2+ dissociates and re-associates, suggesting weaker binding, which is being investigated in silico.
The simulation is consistent with the hypothesis of a unique TnI Ser 22/23-TnC Ca2+-binding loop II interaction that is destabilized by phosphorylation, thus weakening the affinity of the regulatory Ca2+.