Figure 2. Inactivation of RyR1 involves out-of-plane rotation of the central block and rearrangement around the Ca²⁺ activation site.

(A) The high-affinity Ca²⁺-binding site in the CD/CTD interface with density around the Ca²⁺ site contoured at 4σ. Contacts within 2.8 Å from Ca²⁺, as well as additional contact Gln3970-Ser4029 within 3.6 Å, are represented by dashed lines. Channel axis is on the left. During the transition from open to inactivated conformations, the CD/CTD block tilts around the Ca²⁺-binding site such that the protruding fourth helix of the CD (h) and connected CTD tilt inward, while the CD-C′ tilts upward and away from the sarcoplasmic reticulum (SR) membrane – with Gln3970 separating from Ca²⁺ by ~6 Å. Arrows and stationary reference lines illustrate the conformational changes undergone with respect to the panel on the left. The region represented relative to the channel is highlighted with a square in panel (C). See Figure 2—figure supplement 1 for the corresponding space-filling representation. (B) Heat map showing Cα-backbone root mean square deviation (RMSD) (in Å) between domain pairs from respective conformations after aligning them (pre-aligned), in their native conformation (unaligned), and after aligning the protomers through their respective CD (CD-aligned). The RMSD difference between unaligned and pre-aligned represents the change caused by domain relocation. (C) Overlaid structures of the CD-CTD-S6 domains in different conformations; only two protomers shown for clarity. Left: in the transition from RyR1-ACP/Ca²⁺ open (gray) to RyR1-ACP/Ca²⁺ inactivated (colored), the CD-CTD block, ‘connected’ by Ca²⁺ coordination, undergoes an out-of-plane rotation around the Ca²⁺-binding site that pushes S6C′ toward the pore axis, closing the channel. Right: in the transition from RyR1-ACP/Ca²⁺ inactivated (colored) to RyR1-ACP/EGTA (green), Ca²⁺ unbinding disconnects the CD from the CTD. Structures at the bottom right of each panel show the comparison of the CD-CTD-S6C′ of the central block after forcing superimposition of their respective CDs. Residue boundaries for relevant domains are specified. (D) Schematics of the conformational changes from RyR1-ACP/EGTA (closed), to RyR1-ACP/Ca²⁺ open, to RyR1-ACP/Ca²⁺ inactivated. Activation, where Ca²⁺ binds to the high-affinity site, is required prior to inactivation. The CD-protruding fourth helix (3753–3769) is indicated by ‘h.’ Colored arrows indicate the conformational change toward the following structure.

Figure 2—figure supplement 1. Reorganization of the high-affinity Ca²⁺-binding site at the CD/CTD interface under different conditions.

Ca²⁺-binding site in the CD/CTD interface in different conformations with involved residues shown with spheres. Residues Glu3983, Glu3967, Gln3970 (CD), and Thr5001 (CTD), within 2.8 Å from Ca²⁺ in the open state, are shown. In going from RyR1-ACP/Ca²⁺ open to RyR1-ACP/Ca²⁺ inactivated, Gln3970 switches its interaction from Ca²⁺ to Ser4029 (interaction within 3.6 Å). Arrows and stationary reference lines illustrate the conformational changes undergone with respect to the panel on the left. The region represented relative to the channel is highlighted with a square in Figure 2C; fourfold axis is on the left.

Figure 2—figure supplement 2. Rotation axes of the CD of RyR1 among the different conformational transitions.

(A) Reproducibility of the conformation of the ‘central block’ of RyR1-ACP/Ca²⁺ inactivated from two independent datasets (A and B, superimposed), with a root mean square deviation (RMSD) of 0.95 Å over the Cα atoms. The central block comprises the CD, EF hand, U-motif, S6C′, and CTD. (B) Transition from RyR1-ACP/Ca²⁺ open (gray) to RyR1-ACP/Ca²⁺ inactivated (colored) involves an out-of-plane rotation of the CD around the yellow-colored axis. This results in inward tilt in CD-N′ (brown arrows) and upward shift/tilt of CD-C′ and connected EF hand domain (blue arrows). Two orthogonal views are shown, and the fourfold axis on the left panels, in front of the structure, is indicated. (C) Transition from RyR1-ACP/Ca²⁺ inactivated (colored) to RyR1-ACP/EGTA closed (gray) involves out-of-plane rotation of the CD of RyR1 around the magenta-colored axis.

Figure 2—video 1. Ca²⁺-induced transitions in activation and inactivation.

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During activation, Ca²⁺ coordination joins the CD and CTD-S6C′ region, separating the S6 four-helix bundle and opening the channel. High Ca²⁺-induced inactivation promotes out-of-plane rotation of the CD-CTD-S6C′ block. This pushes the S6 helices towards the pore axis, closing the channel. Only two protomers in diagonal are shown.