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

Abstract

Cardiac troponin C (cTnC) is the regulatory protein that initiates cardiac contraction in response to Ca TnC binding Ca initiates a cascade of protein-protein interactions that begins with the opening of the N-terminal domain of cTnC, followed by cTnC binding the troponin I switch peptide (TnI). We have evaluated, through isothermal titration calorimetry and molecular-dynamics simulation, the effect of several clinically relevant mutations (A8V, L29Q, A31S, L48Q, Q50R, and C84Y) on the Ca affinity, structural dynamics, and calculated interaction strengths between cTnC and each of Ca and TnI Surprisingly the Ca affinity measured by isothermal titration calorimetry was only significantly affected by half of these mutations including L48Q, which had a 10-fold higher affinity than WT, and the Q50R and C84Y mutants, each of which had affinities 3-fold higher than wild type. This suggests that Ca affinity of the N-terminal domain of cTnC in isolation is insufficient to explain the pathogenicity of these mutations. Molecular-dynamics simulation was used to evaluate the effects of these mutations on Ca binding, structural dynamics, and TnI interaction independently. Many of the mutations had a pronounced effect on the balance between the open and closed conformations of the TnC molecule, which provides an indirect mechanism for their pathogenic properties. Our data demonstrate that the structural dynamics of the cTnC molecule are key in determining myofilament Ca sensitivity. Our data further suggest that modulation of the structural dynamics is the underlying molecular mechanism for many disease mutations that are far from the regulatory Ca-binding site of cTnC.

Binding of Calcium and Magnesium to Cardiac Troponin C

“…As seen in previous reports, we found the binding of Ca 2+ to N-cTnC to be driven by entropy and unfavorable enthalpy (Table S2) (Stevens, Rayani et al 2017, Johnson, Fulcher et al 2019). The favorable ΔS may be due in part to the hydration enthalpy of Ca 2+ which is thought to be on the order of ~375 kcal*mol -1 and slightly lower than that of Mg 2+ (~460 kcal*mol -1 ) (Smith DW, 1977).…”

“…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.…”

“…An extinction coefficient of 1490 M -1 cm -1 and 4595 M -1 cm -1 , and a molecular weight of 10.1 kDa and 18.4 KDa were used to determine protein concentration for the N-cTnC and full length cTnC constructs, respectively, by 280 nm UV-vis spectroscopy using a NanoDrop 2000 spectrophotometer (Thermo Scientific). The final dialysis buffer was used to dilute the protein samples to a final concentration of 200 µM for the N-terminal construct and 150 µM for full length cTnC as described previously (Stevens, Rayani et al 2017).…”