Modulation of Structure and Dynamics of Cardiac Troponin by Phosphorylation and Mutations Revealed by Molecular Dynamics Simulations

Figures

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  Figure 1. (A) The effects of phosphorylation and the G159D mutation on Ca2+activation of thin filaments. The plots show the decrease in Ca2+ sensitivity upon phosphorylation and the absence of this shift (uncoupling) due to the G159D mutation, measured by an in vitro motility assay. Dotted lines, open points wild type; solid lines and points, G159D; unphosphorylated TnI, squares; phosphorylated TnI, circles. Mean and SEM are shown. The lines are fits of the data to the Hill equation. Replotted from data of Dyer et al.7 (B) Representative structure of the backbone of the unphosphorylated troponin core from molecular dynamics simulation. The residues are colored according to their root mean square fluctuation (RMSF), with the highest RMSF as the deepest red. The locations of the phosphorylatable serines, 22 and 23, in NcTnI (magenta arrow), cTnC G159 (blue arrow), and the switch peptide (green arrow) are indicated. Bound Ca2+ ions are colored in green. The N-terminal domains of troponin C (NcTnC) and of the ITC domain are outlined in blue. The ITC domain contains the C-terminus of TnC (94–161), helices H1 and H2 of cTnI (42–136), and helices H1 and H2 of cTnT (201–277).

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  Figure 2. Heat maps showing the key interactions between troponin peptides related to phosphorylation. The scale represents percentage of interactions observed in 37,500 frames. Selected regions of interest are shown. The complete set of interactions are shown in Supplementary Figure 6. Numerical values for the key interactions are presented in Supporting Information Table 1 with a detailed commentary. (A) Ionic interactions between NcTnI (1–34) and NcTnC (1–84). NcTnI is anchored to NcTnC via Arg9 interacting with NcTnC Gln15 and 19 and Arg 12 interacting with NcTn C Asp25 and Glu32 (orange circle). A strong interaction between NcTnC Lys33 and NcTnI Arg19, 20, and 21 is lost on phosphorylation (blue circle), while interaction with NcTnC Lys39 is gained (green circle). (B) Ionic intramolecular interaction within NcTnI (1–34 vs 1–48). Phosphorylation promotes interaction of phosphoserine 22 with Arg19, 21, and 26 (blue circle). (C–E) Key interactions of CcTnT (C) ionic interactions between CcTnT (280–288) and NcTnC (1–46). There is a medium interaction formed between NcTnC Asp33 and Arg286 upon phosphorylation (blue circle). (D) Ionic interactions between CcTnT (281–288) and NcTnI (1–40).…

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  Figure 3. Representative structures of the cardiac troponin core for wild type and G159D in the unphosphorylated and phosphorylated states. (A, B) Representative structure of wild-type cardiac troponin with surface rendering of NcTnC (white), NcTnI (blue), switch peptide (green), and CcTnT (red). Views are from the side and the “top” of troponin. Rotating models are presented as mp4 movies, and the PDB files used to generate the structures are presented in the supplement. (A) Unphosphorylated, (B) phosphorylated. (C, D) Partial model showing the key phosphorylation-dependent interactions between NcTnI and NcTnC only. TnI Arg19–Arg26 and TnC Asp33, Thr38, and Lys39 are shown in stick format. Rotating models are presented in the supplement. (C) Unphosphorylated, (D) phosphorylated. (E) The interdomain peptide of cTnC in unphosphorylated (cyan) and phosphorylated (blue) states. Phosphorylated and unphosphorylated representative structures are aligned to the IT coiled coil. The interdomain peptide (86–95), helix D, and part of helix E are highlighted to illustrate the lengthening and twisting of the peptide upon phosphorylation. Distribution of interdomain peptide lengths is shown in…

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  Figure 4. (A) Definition of the interdomain angle. Head: the average of C-alpha coordinates of TnC3–85; fulcrum: the average of C-alpha coordinates of TnC94–157; end of IT arm: the average of C-alpha coordinates of TnT241–251 and TnI69–76. (B) Probability distribution of the angle between the NcTnC and ITC domains.

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  Figure 5. (A) Model of the backbone structure of NcTnC showing the relative disposition of helices A and B in the unphosphorylated state. (B) The distribution of the interhelical angle formed by helices A and B in the unphosphorylated and phosphorylated (SEP) states for the wild-type and cTnC G159D-mutant troponin. The definition of open and closed angles is adopted from Lindert et al.36