Structural changes in troponin during activation of skeletal and heart muscle determined in situ by polarised fluorescence

Figures

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  Fig. 1. The cardiac thin filament in the Ca2+-free or APO state (A) and the Ca2+-bound state (B), from Yamada et al. 2020 (PDB 6kn7 and 6kn8, respectively) fitted into the cryo-EM density volumes (EMDB accession codes EMD-0728 and EMD-0729, respectively). Actin is oriented with its pointed end up. The two troponins (Tn1 and Tn2) are non-equivalent and elongated, spanning seven actin monomers and cross-linking tropomyosin (Tm) molecules on opposite actin strands. The N-terminus of troponin T (TnT) crosses over the head-to-tail junction of Tm, which is located farther away from the C-terminal domain of troponin C (TnCC) by one actin subunit in Tn1 as compared to Tn2. In the APO state, the C-terminus of troponin I (TnIC) lies parallel to and binds tropomyosin (Tm) and actin covering myosin binding sites (TnIC lock). In the Ca2+-loaded state, the N-terminal domain of troponin C (TnCN) binds a helical region of TnI150-163 or switch region (TnIswitch), which fits into the exposed hydrophobic pocket in TnCN, thus lifting TnIC from the thin filament (TnIC unlock). A model of TnI160-210 is shown for illustration purposes only as the density for TnI167-210 is absent in the Ca2+-bound state. The…

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  Fig. 2. FISS workflow. A Cartoon representation of the calcium- and switch peptide–bound chicken fast skeletal muscle TnC (sTnC) in lemon green with C-helix coloured in salmon pink (PDB 1YTZ; Vinogradova et al. 2005). Ca2+ ions are shown as black spheres. The chicken fast skeletal switch peptide (sTnIswitch) encompassing residues 115–144 is coloured in slate. Using site-directed mutagenesis, glutamic acid residues at positions 57 and 64 in sTnC (C-helix) are replaced by cysteines. B The recombinant double cysteine mutant TnC is expressed in E. coli and purified (Ferguson et al. 2003). The reactive sulphydryl groups of the cysteines in sTnC are cross-linked with a bifunctional rhodamine dye bis-iodoacetamidorhodamine (BR-I2), and the 1:1 BR-sTnC conjugates are purified to greater than 95% homogeneity. The double arrow indicates the fluorescence dipole, which is parallel to the long axis of the xanthene ring system. C Cartoon representation of the bifunctional rhodamine-labelled sTnC. The N-lobe (sTnCN.2Ca.2+.sTnI116-132.BR57-64; PDB 1NPQ; Mercier et al. 2003) is shown in cyan aligned with sTnC 1YTZ (lemon green). The BR fluorescence dipole is approximately parallel to the C-helix…

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  Fig. 3. Maximum entropy analysis of skeletal muscle FISS data during relaxation and maximum calcium activation. The ME maps were replotted using data from Ferguson et al. (2003) and Knowles et al. (2012). Helices are named from the N-terminus: N, A, B, C, D, E, F and G. A Cartoon representation of the chicken fast skeletal N-lobe in the Ca2+-free or APO state (PDB 1TNP, Gagné et al. 1995). The BR dipoles on helices N (BR6-13), A (BR18-25) and C (BR57-64) are shown in blue, green and salmon pink, respectively. B ME orientation distribution of the skeletal N-lobe domain in relaxed muscle fibres based on the N-, A- and C-helix probe data and the APO NMR structure 24 from 1TNP. The two high-probability orientations are equivalent and represent the two ends of the dipole or the two sarcomere halves. Therefore, only one of them is marked with a black cross (β = 102°, γ = − 30°). This orientation with respect to the thin filament axis (pointed end up) is shown in A. C The orientation distribution of the skeletal N-lobe during active contraction is based on the Ca2+-loaded NMR structure (PDB 1NPQ, sTnCN.2Ca2+.sTnI116-132.BR57-64, Mercier et al. 2003). The black cross indicates the distribution…

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  Fig. 4. Comparison of cardiac and skeletal TnCN structures in different states. The regulatory domain of troponin is viewed along helix D. A–E The Ca2+- and switch peptide–induced structural transitions in the skeletal N-lobe. A Superposition of APO crystal structures of sTnCN along D-helix (EFLVMMVRQ): chicken skeletal TnCN in wheat (PDB 1YV0, Vinogradova et al. 2005), turkey skeletal TnCN in orange (PDB 5TNC, Herzberg and James 1985), rabbit skeletal TnCN in light magenta (PDB 1A2X, Vassylyev et al. 1998). The N-lobe is in a closed conformation with a buried hydrophobic pocket. B Solution NMR family of structures of the APO chicken skeletal TnCN in violet (PDB 1TNP, Gagne et al. 1995). The interhelical angles are similar to the crystal structures, except for the C/D angle, which is more open. C NMR assembly of structures of the Ca2+-loaded chicken skeletal TnCN in deep teal (PDB 1TNQ, Gagne et al. 1995). In skeletal muscle, calcium is sufficient to induce N-lobe opening with the movement of helices B and C away from N, A and D. This exposes a hydrophobic patch to which TnIswitch can bind. D NMR assembly of structures of the Ca2+-and switch peptide–bound chicken skeletal TnCN shown in…

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  Fig. 5. Troponin core domain structures in the Ca2+-bound state. TnC is depicted in grey, TnI in slate and TnT in bright orange. The IT arm in the different Tn complexes is oriented as in the active state according to Model 2 for skeletal Tn (Knowles et al. 2012) and Sevrieva et al. (2014) for cardiac muscle, whereby the thin filament axis is vertical with the pointed end upward as in Fig. 3. The D-helix is depicted in dark grey, and the line indicates that it is at a different angle with respect to the thin filament in the different complexes. A Skeletal troponin structure sTnC5-163.4Ca2+.sTnI4-144.sTnT160-248 (PDB 1YTZ, Vinogradova et al. 2005). The probe dipoles are shown for the N-lobe: N-helix (BR6-13) in blue, A-helix (BR18-25) in green and helix C (BR57-64) in blue. C-lobe probe dipoles: E-helix (BR97-104) in deep purple, F-helix (BR117-124) in lime green, FG interhelix (BR120-136) in yellow and G-helix (BR133-140) in cyan. B Cardiac troponin structure cTnC2-90, 93–161.3Ca2+.cTnI37-138, 149–162.cTnT200-274 (PDB 1J1D, Takeda et al. 2003). N-lobe probes: N-helix (BR3-10) in salmon pink, NA interhelix (BR10-17) in yellow, A-helix (BR15-22) in green, B-helix (BR39-46) in deep teal and…

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  Fig. 6. Maximum entropy analysis of cardiac muscle FISS data during relaxation and maximum calcium activation. The ME maps were replotted using data from Sevrieva et al. (2014). A Cartoon representation of the human cardiac N-lobe in its Ca2+-free or APO R1 orientation (PDB 1SPY, Spyracopoulos et al. 1997; Sevrieva et al. 2014). The BR dipoles on helices N (BR3-10), NA (BR10-17), A (BR15-22), B (BR39-46) and C (BR55-62) are shown in blue, yellow, green, deep teal and salmon pink, respectively. B ME orientation distribution of the cardiac N-lobe domain in relaxed trabeculae based on the N-, NA-, A-, B- and C-helix probe data and the APO NMR average structure (PDB 1SPY). There are two high-probability orientations labelled R1 and R2 (see Table 1). Their equivalent peaks are also shown. The white cross indicates the physiological state-matched orientation in skeletal muscle. Calculated state-matched β and γ from available cryo-EM fitted structures (Table 1): black diamond (Tn1, Yamada et al. 2020), black circle (Tn2, Yamada et al. 2020), cyan diamond (Oda et al. 2020), magenta diamond (U-Tn, Risi et al. 2021b), magenta circle (L-Tn, Risi et al. 2021b). A1 and A3 represent active…

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  Fig. 7. Central sections of maximum entropy maps of cardiac muscle FISS data during relaxation and maximum calcium activation showing the cryo-EM conformations from Risi et al. (2024). The ME maps were replotted using data by Sevrieva et al. (2014). A ME orientation distribution of the cardiac N-lobe domain in relaxed trabeculae. The white cross indicates the physiological state-matched orientation in skeletal muscle. Calculated β and γ from the cryo-EM fitted structures by Risi et al. (2021b) and Risi et al. (2024) (Table 2): magenta diamond (U-Tn, Risi et al. 2021b), magenta circle (L-Tn, Risi et al. 2021b), white diamond (Ca2+-free upper or CF U-Tn, Risi et al. 2024), white circle (Ca2+-free lower or CF L-Tn, Risi et al. 2024), light pink orange dotted diamond (Ca2+-free rotated upper or CF-R U-Tn, Risi et al. 2024), light pink orange dotted circle (Ca2+-free rotated lower or CF-R L-Tn, Risi et al. 2024), cornflower blue diamond (Ca2+-free tilted upper or CF-T U-Tn, Risi et al. 2024) and cornflower blue circle (Ca2+-free tilted lower or CF-T L-Tn, Risi et al. 2024). A1 is the active orientation from the ME map in B. B ME orientation distribution of the cardiac N-lobe domain during…