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 Ca(2+) 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 Ca(2+)-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 Ca(2+) 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 Ca(2+) interaction, whereas the ITC analysis includes the Ca(2+) binding and conformational change of the TnC molecule. ITC analysis has revealed that ssTnC has higher Ca(2+) affinity than cTnC for Ca(2+) 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 Ca(2+) affinity while simultaneously increasing the Ca(2+) sensitivity of the myofilament. We propose that the preferential expression of cTnC at lower temperatures increases myofilament Ca(2+) sensitivity by this mechanism, despite the lower Ca(2+) affinity that we have measured by ITC.

Binding of Calcium and Magnesium to Cardiac Troponin C

“…The interaction of N-cTnC with either Ca 2+ or Mg 2+ was found to be associated with a positive ΔH, so the interaction is driven by entropy (Figure 1) consistent with previously published data (Skowronsky, Schroeter et al 2013, Tanaka, Takahashi et al 2013, Stevens, Rayani et al 2016, Stevens, Rayani et al 2017. This supports our interpretation that the endothermic component between full-length cTnC and Ca 2+ is due to binding to the N-doman.…”

“…Binding of Ca 2+ /Mg 2+ to site II is characterized by an endothermic interaction as indicated by our titrations on the N-terminal domain in this and previous publications (Stevens, Rayani et al 2016, Stevens, Rayani et al 2017. From this, and ITC work on full-length cTnC by others (Johnson, Fulcher et al 2019), we can deduce that the exothermic interactions seen above (Figure 5) result from interactions with site III/IV.…”

“…The D76A/D73A construct was made using site-directed mutagenesis carried out by GenScript (New Jersey, USA). Expression and purification of all constructs were carried out as described previously (Stevens, Rayani et al 2016, Stevens, Rayani et al 2017. In brief, 100 mL of lysogeny broth (LB) was supplemented with 50 μg/mL ampicillin and a glycerol stock stab and grown over-night at a shaking speed of 250 rpm and 37ºC.…”

Changes in the dynamics of the cardiac troponin C molecule explain the effects of Ca2+-sensitizing mutations

“…A stop codon was introduced at position 90 by following the Phusion site-directed mutagenesis protocol (Thermo). Protein expression, purification, melting point determination, and ITC were carried out using protocols modified from previous work (30). The N-cTnC construct corresponds to the human TNNC1 gene (Uniprot ID P63316), which was cloned into the pET-21aϩ expression vector (Novagen).…”

“…The structural effects of Ca 2ϩ binding have been examined through NMR and X-ray crystallographic data (22)(23)(24)(25), including for the HCM-associated TnC mutant L29Q (24,26). These structures and data from MD simulations have demonstrated minimal effects of sequence substitutions on the static structure but a greater effect on the dynamics of the protein (27)(28)(29)(30).…”