Glutamate 139 of tropomyosin is critical for cardiac thin filament blocked-state stabilization

Abstract

The cardiac thin filament proteins troponin and tropomyosin control actomyosin formation and thus cardiac contractility. Calcium binding to troponin changes tropomyosin position along the thin filament, allowing myosin head binding to actin required for heart muscle contraction. The thin filament regulatory proteins are hot spots for genetic mutations causing heart muscle dysfunction. While much of the thin filament structure has been characterized, critical regions of troponin and tropomyosin involved in triggering conformational changes remain unresolved. A poorly resolved region, helix-4 (H₄) of troponin I, is thought to stabilize tropomyosin in a position on actin that blocks actomyosin interactions at low calcium concentrations during muscle relaxation. We have proposed that contact between glutamate 139 on tropomyosin and positively charged residues on H₄ leads to blocking-state stabilization. In this study, we attempted to disrupt these interactions by replacing E139 with lysine (E139K) to define the importance of this residue in thin filament regulation. Comparison of mutant and wild-type tropomyosin was carried out using in-vitro motility assays, actin co-sedimentation, and molecular dynamics simulations to determine perturbations in troponin-tropomyosin function caused by the tropomyosin mutation. Motility assays revealed that mutant thin filaments moved at higher velocity at low calcium with increased calcium sensitivity demonstrating that tropomyosin residue 139 is vital for proper tropomyosin-mediated inhibition during relaxation. Similarly, molecular dynamic simulations revealed a mutation-induced decrease in interaction energy between tropomyosin-E139K and troponin I (R170 and K174). These results suggest that salt-bridge stabilization of tropomyosin position by troponin IH₄ is essential to prevent actomyosin interactions during cardiac muscle relaxation.

Keywords: Blocked-state stabilization; Cardiac muscle regulation; Helix-4; In vitro motility; Tropomyosin; Troponin.

Fig. 1.. Predicted interactions between Tpm 139 and Helix-4 of TnI.

Ribbon diagram of the H₄ of TnI (cyan) and Tpm (magenta) showing the electrostatic interaction between glutamate 139 on Tpm (red spheres) and arginine 170 and lysine 174 on TnI (blue spheres). Actin is a grey ribbon. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2.. E139K Tpm increases Ca⁺² sensitivity

Both graphs were fit to the Hill equation given by $Y=\mathit{O}\mathit{f}\mathit{f}\mathit{s}\mathit{e}\mathit{t}+(\left[{\mathit{C}\mathit{a}}^{\mathit{2}+}\right]n_H)\times (V_{\mathit{m}\mathit{a}\mathit{x}}-\mathit{O}\mathit{f}\mathit{f}\mathit{s}\mathit{e}\mathit{t})/(\left[{\mathit{C}\mathit{a}}^{\mathit{2}+}\right]n_H+{pCa}_{50}{n}_H)$, where $n_H$ is the Hill coefficient. (A) Sliding speed of fluorescently labeled actin with bound Tn and either WT (black) or E139K Tpm (magenta) was measured with increasing calcium concentration. Fit parameters for panel A: $p{Ca}_{\mathit{50}}$ (WT: 5.80 ± 0.23, E139K: 6.38 ± 0.13), $Offset$ (WT: 0.02 ± 0.35, E139K: 1.03 ± 0.30), $n_H$ (WT: 0.5189 ± 0.1139, E139K: 0.6739 ± 0.1488), and $V_{max}$ (WT: 8.80 μm*sec⁻¹ ± 0.84and E139K: 8.06 μm*sec⁻¹ ± 0.39) (B) Percent of motile filaments vs calcium concentration. Fit parameters for panel B: $p{Ca}_{\mathit{50}}$ (WT: 7.04 ± 0.5, E139K: 7.563 ± 0.8), $Offset$(WT: 4.40 ± 7.80, E139K: 17.33 ± 15.90), $n_H$ (WT: 0.75 ± 0.64, E139K: 0.46 ± 0.98), and Max % Motile (WT: 91.55% ± 10.28 and E139K: 85.43% ± 7.39). Assays were performed on 5 separate days using 2 independent Tpm preps and averaged together with standard error of the mean represented by the error bars. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3.. Tpm intramolecular salt bridge-induced hydrophobic core gap stability.

Comparison of the solvent accessible surface for a representative frame during the last 10 ns of MD simulation for the WT (grey, top row) and E139K (magenta, bottom row) Tpm in the B-state, C-state, and actin-free simulations. The surface over residues 139 and 142 was made transparent to show the proximity of the side chains. Note the larger hole between the chains formed in the mutant simulations. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4.. E139K Tpm diminished TnI H₄ contacts.

(A) F-actin was incubated with varying concentrations of Tpm (0.1 μM – 3.5 μM) in high salt binding buffer (15 mM BES, 5 mM MgCl₂, 200 mM NaCl, pH 7.0) to reach equilibrium binding and spun down with nearly all F-actin pelleting along with any bound Tpm. This data was quantified to produce a curve fit to ‘specific binding with Hill slope’ $(Y=B_{\mathit{m}\mathit{a}\mathit{x}}\mathrm{*}{X}^h/\left(K_{\mathit{d}}^h+X^h\right)$ with $B_{max}$ being the max fraction saturated, Kd being the equilibrium constant, and h representing the hill slope of the fit. The equilibrium constant ($K_d$) of WT-Tpm and Tpm-E139K was 0.21 ± 1.46 μM and 0.27 μM ± 0.42, respectively. Hill coefficient values ($h$) were 0.90 ± 0.85 for WT and 1.09 ± 0.67 for E139K. $B_{max}$ for WT was 1.16 ± 1.2 and for E139K 1.07 ± 0.51. The data points are plotted from 4 separate experiments (n = 4) on separate days. $K_d$ and respective curve fit between WT and E139K were calculated based on a 95% confidence interval (p < 0.05). (B–D) Calculated electrostatic interaction energies of individual Tpm residues with actin in Tpm’s B-state (B) or C-state (C) positions or with TnI in the B-state (D). In (E), the electrostatic interaction energy by residue of TnI to Tpm is shown. Values were calculated as an average over 100 frames from the last 10 ns of MD simulation. The asterisks indicate the mutation site on Tpm, residue 139, (D) and the corresponding predicted interaction sites on TnI, residues 170 and 174 (E).