Molecular Dynamics Simulations of the Cardiac Ryanodine Receptor Type 2 (RyR2) Gating Mechanism

Figures

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  Figure 1. Computational model of the RyR2 system. A computational model of the closed (4C) RyR2 system containing a portion of the central domain (residues 4099–4206) and the transmembrane channel domain (residues 4485–4963) is shown. Key structural elements such as the U-motif (residues 4167–4184, black), the S4S5L (residues 4746–4766, green), and the S6 helix (residues 4839–4889, blue) are highlighted in the first subunit (gray). The extent of the membrane region is roughly indicated by two horizontal black lines, and the cytosolic and luminal regions are indicated by C and L, respectively. This image of the channel is an average structure taken over the last 100 ns of a 1 μs MD simulation. Water molecules, DPPC lipids, and counterions have been omitted from this image for clarity. The image of the RyR2 channel was produced using UCSF Chimera X 1.4.

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  Figure 2. Initial alignment of the first closed RyR2 subunit with the first open RyR2 subunit. An alignment from the initial models of the first subunit in the closed 4C RyR2 model system (tan) and the first subunit in the open 4O RyR2 model system (light blue) before minimization and MD simulations were carried out. The S4S5L (residues 4746–4766) is highlighted for the closed RyR2 model system (blue) and for the open RyR2 model system (red). The alignment of the two subunits was performed using UCSF Chimera 1.15, while the images were visualized using UCSF Chimera X 1.4.

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  Figure 3. Pore radius profiles of RyR2 systems. A numerical plot of the pore radius as a function of the z-coordinate is provided for the structures of the eight RyR2 model systems. The single structure analyzed in these plots was obtained by averaging across 100 snapshots sampled every 1 ns over the last 100 ns of the MD simulation. The pore radius profiles of the original closed 4C and open 4O systems are given in panel (A), the pore radius profiles of the chimera systems 1C3O, 1C3O-open-S4S5L, and 1C3O-H4762P are given in panel (B), the pore radius profile of the chimera system 1C3O-HID is given in panel (C) along with the 1C3O system for ease of comparison, and the pore radius profiles of chimera systems 1C3O-open-S4S5L-HID and 1C3O-open-S4S5L-H4762P are given in panel (D) along with the 1C3O-open-S4S5L system for ease of comparison. To facilitate comparisons between the various systems, the origin of the z-axis was adjusted for all eight systems so that the zero point corresponded to the hydrophobic gate. Positive z-values indicate the luminal side of the channel and negative values appear on the cytosolic side. Visualizations of the channel and pore for these plots are available in…

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  Figure 4. Minimum pore radius of RyR2 systems. A comparison of the minimum pore radius is provided for all eight RyR2 model systems. For the three closed-state systems (4C, 1C3O, and 1C3O-HID) and one open-state system (1C3O-open-S4S5L-H4762P), the minimum pore radius corresponded to the hydrophobic gate formed by four I4867 residues. For the remaining four open-state systems (4O, 1C3O-open-S4S5L, 1C3O-open-S4S5L-HID, and 1C3O-H4762P), the minimum pore radius is found within the luminal domain, between z = 0 and 25 Å (see Figure 3).

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  Figure 5. Number of S4S5L high percent occupancy interactions observed in the closed and open RyR2 systems. The number of high percent occupancy interactions involving the S4S5L (residues 4746–4766) observed by residue number within our three closed (4C, 1C3O, and 1C3O-HID) and three open (4O, 1C3O-open-S4S5L, and 1C3O-open-S4S5L-HID) RyR2 systems is given. The number of interactions at a given residue position within the S4S5L is given in panel (A), while the number of interactions at each residue position that the S4S5L interacted with is given in panel (B). In producing this figure, we pooled data from all four subunits in the three closed RyR2 systems (4C, 1C3O, and 1C3O-HID) and compared it to the pooled data from all four subunits in three open RyR2 systems (4O, 1C3O-open-S4S5L, and 1C3O-open-S4S5L-HID). In panel (B), residue numbers for regions within the U-motif, S4S5L, and the S6 helix that we focus on in our discussion are highlighted along the x-axis. The full set of high percent occupancy interaction data is available in Tables S1 and S2.

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  Figure 6. High percent occupancy S4S5L interactions in the 4C and 4O systems. S4S5L residues involved in high percent occupancy interactions are given for 4C (A) and 4O (B). The S4S5L (residues 4746–4766) is colored green, with H4762 colored orange. A portion of the S6 helix (residues 4859–4869) is colored purple, with I4867 colored yellow. Key interactions outside of these two regions are colored magenta when they appear on subunit 1. A portion of the adjacent subunit 2 (residues 4869–4879) is given in gray, while high percent occupancy interactions with subunit 2 are labeled (s2) for clarity. Both structures for 4C and 4O represent an average structure over the last 100 ns of the 1 μs MD simulation. This image was produced using UCSF Chimera X 1.4.

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  Figure 7. Number of inter-subunit high percent occupancy interactions observed in the closed and open RyR2 systems. The number of inter-subunit high percent occupancy interactions (>30% occupancy) listed by residue number that were observed within our three closed (4C, 1C3O, and 1C3O-HID) and three open (4O, 1C3O-open-S4S5L, and 1C3O-open-S4S5L-HID) RyR2 systems is given. To limit the number of interactions along the x-axis for this plot, an additional restriction was made where only residue locations where either the closed or open systems had an interaction appear at least three times were included in this plot. In producing this figure, we pooled data from all four subunits in the three closed RyR2 systems (4C, 1C3O, and 1C3O-HID) and compared it to the pooled data from all four subunits in three open RyR2 systems (4O, 1C3O-open-S4S5L, and 1C3O-open-S4S5L-HID). Residue numbers for regions within the U-motif, S4S5L, and the S6 helix that we focus on in our discussion are highlighted along the x-axis. The full set of high percent occupancy interaction data is available in Tables S3 and S4.

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  Figure 8. Residue N4763 on the S4S5L can act as a switch that controls RyR2 channel gating. N4763 (green spheres) within the S4S5L (residues 4746–4766, green ribbons) in subunit 1 is normally out of position in the closed 4C system in panel (A) to interfere with an inter-subunit interaction between E4872 (blue spheres) on the S6 helix in subunit 1 (residues 4839–4889, blue ribbons) and R4874 (orange spheres) on the S6 helix in subunit 2 (residues 4839–4889, orange ribbons). In the open 4O system in panel (B), a shift in the position of the S4S5L allows N4763 to interact with R4874, replacing the previous inter-subunit interaction between R4874 and E4872. Both structures for 4C and 4O represent an average structure over the last 100 ns of the 1 μs MD simulation. This image was produced using UCSF Chimera X 1.4.

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  Figure 9. Key high percent occupancy interactions between the S4S5L and the U-motif. A closeup of the key high percent occupancy interacting residues (solid spheres) between the S4S5L (residues 4746–4766, green) and the U-motif (residues 4167–4184, black) is provided for the closed 4C system in panel (A) and the open 4O system in panel (B). Residue E4932, which is one of a trio of residues that coordinate to Ca2+ at the calcium activation site, is shown with red spheres. Both structures for 4C and 4O represent an average structure over the last 100 ns of the 1 μs MD simulation. This image was produced using UCSF Chimera X 1.4.

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  Figure 10. Conformation of the four-subunit S4S5L structure in the 1C3O chimera RyR2 system indicates cooperative channel closing. The structure in the vicinity of the hydrophobic gate in the transmembrane domain of the RyR2 is shown for the closed 4C system (A), the open 4O system (B), the closed 1C3O chimera system (C), and the 1C3O-open-S4S5L chimera system (D). The isoleucine residue that forms the hydrophobic center of the channel pore (I4867) is depicted using orange VDW spheres for the first subunit and purple VDW spheres for the other three subunits. The S4S5L (residues 4746–4766) is colored blue in the first subunit of each system while the S4S5L on the other three subunits is colored green. All images represent an average structure over the last 100 ns of the 1 μs MD simulation. This image was produced using UCSF Chimera X 1.4.