Characterization of Zebrafish Cardiac and Slow Skeletal Troponin C Paralogs by MD Simulation and ITC

Abstract

Zebrafish, as a model for teleost fish, have two paralogous troponin C (TnC) genes that are expressed in the heart differentially in response to temperature acclimation. Upon Ca2+ binding, TnC changes conformation and exposes a hydrophobic patch that interacts with troponin I and initiates cardiac muscle contraction. Teleost-specific TnC paralogs have not yet been functionally characterized. In this study we have modeled the structures of the paralogs using molecular dynamics simulations at 18°C and 28°C and calculated the different Ca2+-binding properties between the teleost cardiac (cTnC or TnC1a) and slow-skeletal (ssTnC or TnC1b) paralogs through potential-of-mean-force calculations. These values are compared with thermodynamic binding properties obtained through isothermal titration calorimetry (ITC). The modeled structures of each of the paralogs are similar at each temperature, with the exception of helix C, which flanks the Ca2+ binding site; this region is also home to paralog-specific sequence substitutions that we predict have an influence on protein function. The short timescale of the potential-of-mean-force calculation precludes the inclusion of the conformational change on the ΔG of Ca2+ interaction, whereas the ITC analysis includes the Ca2+ binding and conformational change of the TnC molecule. ITC analysis has revealed that ssTnC has higher Ca2+ affinity than cTnC for Ca2+ overall, whereas each of the paralogs has increased affinity at 28°C compared to 18°C. Microsecond-timescale simulations have calculated that the cTnC paralog transitions from the closed to the open state more readily than the ssTnC paralog, an unfavorable transition that would decrease the ITC-derived Ca2+ affinity while simultaneously increasing the Ca2+ sensitivity of the myofilament. We propose that the preferential expression of cTnC at lower temperatures increases myofilament Ca2+ sensitivity by this mechanism, despite the lower Ca2+ affinity that we have measured by ITC.