Abstract
During β-adrenergic stimulation, cardiac troponin I (cTnI) is phosphorylated by protein kinase A (PKA) at sites S23/S24, located at the N-terminus of cTnI. This phosphorylation has been shown to decrease KCa and pCa50, and weaken the cTnC-cTnI (C-I) interaction. We recently reported that phosphorylation results in an increase in the rate of early, slow phase of relaxation (kREL,slow) and a decrease in its duration (tREL,slow), which speeds up the overall relaxation. However, as the N-terminus of cTnI (residues 1–40) has not been resolved in the whole cardiac troponin (cTn) structure, little is known about the molecular-level behavior within the whole cTn complex upon phosphorylation of the S23/S24 residues of cTnI that results in these changes in function. In this study, we built up the cTn complex structure (including residues cTnC 1–161, cTnI 1–172, and cTnT 236–285) with the N-terminus of cTnI. We performed molecular-dynamics (MD) simulations to elucidate the structural basis of PKA phosphorylation-induced changes in cTn structure and Ca2+ binding. We found that introducing two phosphomimic mutations into sites S23/S24 had no significant effect on the coordinating residues of Ca2+ binding site II. However, the overall fluctuation of cTn was increased and the C-I interaction was altered relative to the wild-type model. The most significant changes involved interactions with the N-terminus of cTnI. Interestingly, the phosphomimic mutations led to the formation of intrasubunit interactions between the N-terminus and the inhibitory peptide of cTnI. This may result in altered interactions with cTnC and could explain the increased rate and decreased duration of slow-phase relaxation seen in myofibrils.
Highlights
Troponin (Tn) and tropomyosin (Tm) regulate thin-filament interactions with the thick filament in a Ca2þ-dependent manner [1,2]
A large change was observed in N-terminal portion of cTnC (NcTnC) and an even more dramatic change was observed for NcTnI
Our goal in this study was to elucidate how the phosphomimic mutant of cardiac troponin I (cTnI) induces changes in the Cardiac Tn (cTn) structure that underlie changes in function reported in the literature [4,5,8,29]
Summary
Troponin (Tn) and tropomyosin (Tm) regulate thin-filament interactions with the thick filament in a Ca2þ-dependent manner [1,2]. Cardiac Tn (cTn) serves as a critical regulator of contraction in cardiac muscle and consists of three distinct subunits (cTnC, cTnI, and cTnT), each named according to their function [3]. Contractile activation of cardiac muscle is initiated by Ca2þ binding to the regulatory domain of cTnC (site II). The resulting conformational change triggers the movement of the switch peptide of the inhibitory subunit cTnI (residues 147–163) toward hydrophobic residues exposed within the N-terminal portion of cTnC (NcTnC). The movement of the cTnI switch peptide pulls the adjacent inhibitory peptide (residues 137–146) away from the actin-Tm complex. The mobility of Tm on the thin filament is increased, resulting in increased exposure of myosin-binding sites on actin
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