Myosin Filaments of Vertebrate Skeletal and Cardiac Muscle are Highly Similar, but not Identical

Figures

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  Figure 1.. Single-particle 3D reconstruction of the vertebrate skeletal C-zone. Color scheme shown at the bottom is retained in all figures. (A) 3D reconstruction of seven crowns of heads showing three types of myosin head arrangements (IHM-C, IHM-D, and IHM-S), fast skeletal myosin binding protein C (fMyBP-C), and titin. (B) The middle five-crown segment of the seven-crown map from panel A after post-processing using DeepEMhancer, segmented to identify the myosin heads and tails, fMyBP-C molecules (orange), and two titin strands (Titin-C, bright green; Titin-M, dark green). (C) Side view of an asymmetric unit of the reconstruction, oriented to optimize visibility of all components. fMyBP-C domains C5–C10 and titin-M and titin-C super-repeat domains 1–11 are numbered. (D) Structure of IHMs, with their two light chains. ELC, essential light chain (pink), RLC, regulatory light chain (purple). (E) The structures of the three IHM types of the cardiac thick filament, CrH (horizontal), CrD (disordered), and CrT (tilted), are depicted in green, red, and yellow, respectively (compare with Fig. 1D). (F) Atomic models of cardiac IHMs (EMD-15353) were docked into our skeletal IHMs’ 3-D density maps to…

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  Figure 2 |. Myosin tail network in skeletal thick filament C-zone. (A) The generated 19 crown map needed to show complete myosin tail layers illustrating the intricate interaction network within the filament C-zone. (B) Cross-sectional slices of the reconstruction (viewed toward the M-line) showing the organization of the myosin tails at crowns IHM-D, IHM-C, and IHM-S. Black lines indicate the association of individual tails with the separate myosin tail layers. (C-E) Density maps show segmented myosin tails and heads from the three distinct crowns axially offset by 430Å that form the myosin tail layers. The two views have an azimuthal 90° separation. They are designated as (C) IHM-S, (D) IHM-C, and (E) IHM-D. Note that the proximal S2 part of the myosin tail is almost three crowns in length in IHM-S and IHM-C whereas that of IHM-D is only about one crown in length. These patterns are similar to those found in cardiac thick filaments. (F) Fitting the three segmented tails of IHM-C, IHM-S, and IHM-D into the cardiac myosin tails from the human cardiac map model (EMD_40468). See also Supplementary Video 2.

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  Fig. 3 |. Titin architecture and domain organization within the C-zone. (A) A pair of eleven-domain titin-C (bright green) and titin-M (dark green) C-zone repeats which align precisely with the arrangement of the three myosin IHMs: IHM-C, -S, and -D. (B) A segmented titin 430Å super-repeat is shown with its domains labeled. While most of the configuration of titin-C and titin-M is similar, their T4-T7 domains differ significantly. (C) To highlight the similarities and differences between cardiac and skeletal titins, we fitted the EM model (left) and atomic structure of cardiac titin (Titin A: ochre, Titin B: yellow; right) from PDB 8G4L into our map. The fit is almost exact resulting in a global change in the shade of green for the two skeletal titins. (D) As in B, the two titin strands adopt distinct conformations; in the T4–T7 region, Titin-C bends away from the Titin-M path toward the myosin backbone to contact the proximal S2 segment and motor domain of IHM-D. Brackets mark the region of different titin conformations. (E) While the IHM-D signal is weak due to its disordered nature, a clear free head myosin density (light blue) is observed at the T6 domain of Titin-C (light green). This…

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  Fig. 4 |. Titin-Myosin interfaces dictate thick filament organization. (A). Density map showing one pair of titin strands (titin-C: bright green, and titin-M: dark green) and myosin molecules IHM-D, IHM-C, and IHM-S, to show their molecular organization relative to the titins. (B-G) Detailed views of all interactions between individual titin strands and myosin tails. T-numbers indicate the individual titin domains involved (T1-T11) and numbers alone indicate crown portion of myosin tails (crowns 1, 3, and 6, counted distally). (B) IHM-D tail binds to two repeats of the T5-T8 domains of Titin-C (Ig–Fn–Fn–Fn), which are in a distinct conformation relative to Titin-M (Fig. 3B). (C) IHM-D tail does not interact with Titin-M. (D) IHM-S tail does not interact with Titin-C. (E) IHM-S interacts minimally (T2, T9, and T10 domains) with Titin-M, showing a weak but present interaction. (F) Only the LMM segment of IHM-C interacts with Titin-C. (G) IHM-C tail extensively interacts with titin-M. T7 and T8 are the only two domains of Titin-M that do not bind to proximal S2 of the tail of IHM-C.

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  Fig. 5 |. MyBP-C engages IHM-D tail, IHM-S, and IHM-C heads (A) 11-crown map provides a comprehensive look at the organization of fMyBP-C (Orange), showing its placement at 430 Å intervals within one segment of the thick filament C-zone. (B) The map further details the interactions between fMyBP-C and myosin molecules (enclosed in a box), specifically highlighting contacts with both the IHM-S and IHM-C heads as well as a sheet of tails derived from three crowns of IHM-D (crowns 3, 6, and 9). (C-D) Magnified views of the map reveal the interactions occurring between the C8-C10 domains and the IHM-D tails. (E) The density of fMyBP-C (colored yellow). (F) The density for domains C10 to C5 of cardiac MyBP-C (colored red) is fitted into our map. (G-H) Highlight similarities and differences between cMyBP-C and fMyBP-C, the map and atomic structure of cardiac MyBP-C (red) are fitted into the fMyBP-C map (orange). All cardiac MyBP-C maps and atomic models referenced above are sourced from PDB 8G4L. (I-J) At a lower contour threshold transverse view and side view a significant density for domain C5 is revealed that is not observed at a higher-contour cut-off; C5 appears to bind to the T8 domain of…