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 Ca²⁺ TnC binding Ca²⁺ 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_SW). 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 Ca²⁺ affinity, structural dynamics, and calculated interaction strengths between cTnC and each of Ca²⁺ and TnI_SW Surprisingly the Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺-binding site of cTnC.

Keywords: calcium; cardiomyopathy; isothermal titration calorimetry (ITC); molecular dynamics; troponin.

Figure 1.

A, structure of the core domain of the Tn complex (PDB code 1J1E) with each of the residues that were selected for this study highlighted. The troponin complex proteins are colored white (cTnC), gray (cTnT), and black (cTnI). B, the isolated N-cTnC domain bound to Ca²⁺ used in the PMF simulations. Helices and mutation sites are labeled. C, the N-cTnC domain bound to the TnI_SW and Ca²⁺ is the system used in the TnI_SW binding simulations. D, a schematic of the N-cTnC construct. The helices, Ca²⁺-binding loop, and the residues being examined in this study are labeled.

Figure 2.

Representative isotherms for each of the TnC constructs at 25 °C. The isotherms for WT and each mutant are endothermic, except for the exothermic L48Q isotherm.

Figure 3.

Structural changes induced by each of the mutations. In each panel, the left side contains a representative structure of each mutation superimposed with the wild-type structure (white). To orient the reader, a red arrow indicates the location of the mutation on the structure. Changes to side chain packing in the immediate area of each mutation are demonstrated on the right for each of the mutations. Although the changes to the backbone are very subtle, there are side chain rearrangements in the local vicinity of the mutations, particularly for the mutations that occur at helix–helix interfaces such as A8V, L48Q, and Q50R.

Figure 4.

The A/B interhelical angle is plotted as a function of time for five replicated simulations of each mutated model. Plots are a rolling average of 250 ps. An interhelical angle less than 110° is considered open, and above 130° is considered closed. There is little difference between WT and most of the constructs, with the exception of the L48Q model. The large angle values in one replicate of the L29Q simulations is an artifact caused by a transient loss and recovery of secondary structure in one of the replicated simulations; the hydrophobic solvent-accessible surface is not increased as a function of this change. The h-sasa as a function of time is reported in supplemental Fig. S4.

Figure 5.

Violin plot demonstrating the distribution of open and closed N-cTnC structures observed over five replicated 1-μs simulations. The open conformation was defined by an AB interhelical angle less than 110°, whereas the closed conformation was defined by an AB interhelical angle greater than 130°. The proportion of open frames is not correlated with the proportion of closed frames. The L48Q construct has the most frames in the open conformation, whereas the A8V is the least closed. This suggests that the destabilization of the closed conformation does not necessarily imply that the open conformation is stabilized.

Figure 6.

Average distance between cTnC and cTnI residues. The mutated TnC residue in each plot is indicated by a gray bar. The structures to the right are representative structures of independent simulations and indicate the differences in the orientation and variability of the cTnI_SW peptide across replicates for each mutant. The TnI_SW is colored as a spectrum from blue at the N terminus to red at the C terminus. The calculated ΔG of interaction is maintained across mutations despite differences in the interaction distance profiles, which suggests a nonspecific interaction. The A31S mutant has a ΔG of interaction with the TnI_SW ∼25% lower than WT (Table 2), perhaps because of shorter interaction distances with the N-terminal region of N-cTnC but longer interaction distances in the vicinity of the A31S mutation and C-terminal portion of the TnI_SW.

Figure 7.

The potential of mean force profile of each of the mutated constructs as a function of center of mass distance between the TnC molecule and Ca²⁺ ion. Each of the mutated constructs has an increased ΔG of Ca²⁺ interaction. L48Q (−32 ± 3 kJ·mol⁻¹) is the closest to WT (−32 ± 4 kJ·mol⁻¹), followed by Q50R (−41.3 ± 3 kJ·mol⁻¹), L29Q (−46 ± 2 kJ·mol⁻¹), C84Y (−46 ± 3 kJ·mol⁻¹⁾ A31S (−46 ± 2 kJ·mol⁻¹), and A8V (−46 ± 5 kJ·mol⁻¹), which are similar to each other.

Figure 8.

Schematic of the energetic landscape of N-cTnC activation. N-cTnC is shown as a cartoon. Ca²⁺ is a blue circle, and the TnI switch peptide is represented as a red ellipse. Lower energy states are more favorable. The orange arrows represent the resistance to the conformational change caused by the hydrophobic cleft. The blue arrows indicate conformational strain introduced by Ca²⁺ binding. The Ca²⁺-bound, open conformation relieves the conformational strain while occluding the hydrophobic cleft and is therefore the most favorable conformation. Mutations that affect the relative stabilities of these states will modify the probability of transitions between them and increase or decrease the Ca²⁺ sensitivity of the myofilament.