Abnormal myosin post‐translational modifications and ATP turnover time associated with human congenital myopathy‐related RYR1 mutations

Figures

• 

  FIGURE 1. Disrupted β/slow myosin heavy chain (MyHC) post‐translational modifications. (A–D) depict four significant differences observed in acetylation and phosphorylation. They show the z‐score for each post‐translational modification. Dots are individual subject's average data. Means and standard deviations also appear on histograms. *denotes a difference (p < 0.05) between controls (CTL) and RYR1 patients (RYR1‐CM) or patients with RYR1 mutations and rhabdomyolysis/myalgia but without any weakness (RYR1‐RM). (E) represents myosin II skip 2 (RCSB PDB:4XA3) with Ser1362‐P and Tyr1375‐P in green. (F) is the myosin II dimer in its closed conformation (10S myosin II, RCSB PDB:6XE9 aligned with MYH7 using BLAST). The SH3 domain is shown in green, while myosin regulatory and essential light chains are displayed in gray. Lys1410‐Ac appears in red.

• 

  FIGURE 2. Abnormal post‐translational modifications on type IIa myosin heavy chain (MyHC). (A–H) show the eight significant differences found in acetylation and phosphorylation. They depict the z‐score for each acetylation. Dots are individual subject's average data. Means and standard deviations also appear on histograms. *denotes a difference (p < 0.05) between controls (CTL) and RYR1 patients (RYR1‐CM) or patients with RYR1 mutations and rhabdomyolysis/myalgia but without any weakness (RYR1‐RM). (I) is the myosin II dimer in its closed conformation (10S myosin II, RCSB PDB:6XE9 aligned with MYH2 using BLAST). The SH3 domain is in green, myosin regulatory and essential light chains are in gray, and the converter region is in pink. Lys35‐Ac, Lys87‐Ac, Lys663‐Ac, and Lys763‐Ac are represented in red. (J) is a schematic view of myosin II skip 1 (RCSB PDB: 4XA1) with Lys1171‐Ac in green.

• 

  FIGURE 3. No effect of myosin heavy chain types (MyHCs) on proportions of myosin relaxed states and their ATP turnover lifetime time. The proportions of myosin heads in the disordered–relaxed (A, P1) and super‐relaxed states (B, P2), as well as their respective ATP turnover lifetimes (C, T1 and D, T2), are depicted. Dots are individual control muscle fibres' data. Means and standard deviations for each fibre type (β/slow or type II MyHC) also appear on histograms.

• 

  FIGURE 4. Increased ATP turnover lifetime of myosin disordered–relaxed state. (A) The proportions of β/slow and type II myosin heavy chain (MyHC) isoforms for controls (CTL), RYR1 myopathic patients (RYR1‐CM), and patients with RYR1 mutations and rhabdomyolysis/myalgia but without any weakness (RYR1‐RM) are presented. The proportions of myosin heads in the disordered–relaxed (B, P1) and super‐relaxed states (C, P2), as well as their respective ATP turnover lifetimes (D, T1 and E, T2), are shown. Dots are individual subject's average data. Means and standard deviations for each subject also appear on histograms. *denotes a difference (p < 0.05) between RYR1‐CM and CTL or RYR1‐RM.

• 

  FIGURE 5. Changes in myosin flexibility and secondary structure with Ser1362‐P and Tyr1375‐P. (A) Cartoon representation of the skip 2 segment (PDB ID: 4XA3, after energy minimization). The positions of Ser1362 and Tyr1375 are each highlighted with red spheres (Cα atoms). (B, C) Root‐mean‐square fluctuation (RMSF) profiles (B) and α‐helix persistence (C) for molecular dynamics (MD) simulations with no post‐translational modifications (gray), with phosphorylated Ser1362 on both chains (Ser1362‐P, green) and with phosphorylated Tyr1375 on both chains (Tyr1375‐P, light blue). For each residue, values are calculated as averages over 7 MD replicas. The α‐helix persistence of a residue is calculated as the percentage of frames in which the residue is found to be part of an α‐helix. The positions of Ser1362 and Tyr1375 in the plots are highlighted with vertical lines.

• 

  FIGURE 6. Alterations of interchain distance and interactions with Ser1362‐P and Tyr1375‐P. (A) Time evolution of the distance between the Cα atoms of Ser1362 (top row) and Tyr1375 (bottom row) in the two chains for no post‐translational modifications (gray), Ser1362‐P (green), and Tyr1375‐P (light blue) molecular dynamics (MD) simulations of the skip 2 segment. The average calculated over all the replicas is plotted in red. (B) Histogram of the number of interchain hydrogen bonds between Tyr1375 and any residue in the opposing chain for chain A (left plot) and chain B (right plot). Histograms show values from all the no PTM (gray) and Tyr1375‐P (light blue) MD replicas. (C) Cartoon representation of the representatives of the two most populated clusters for no PTM (left panels) and Tyr1375‐P (right panels) MD simulations. Tyr1375(‐P) residues are shown as sticks, together with the amino acids that form hydrogen bond interactions with the Tyr1375(‐P) side chains. Cluster populations are reported in brackets. Hydrogen bonds detected by VMD are shown as dashed green lines.