Molecular basis for allosteric regulation of the type 2 ryanodine receptor channel gating by key modulators

Fig. 1.. PCB95 facilitates Ca2+ to overcome the barrier in opening the gate of RyR2. (A) Domain organization of a RyR2 protomer. To facilitate description of the conformational changes throughout this study, the domains with resolved secondary structure elements are divided into 3 layers from the luminal side to the cytoplasmic side. Layer 1 contains the channel domain. Layer 2 contains the central domain. Layer 3 contains the HD1 and handle domain and NTD-C. (B) Ca2+ induces conformational changes in the channel domain but is insufficient to open the channel (Left). Addition of PCB95 induces further conformational changes that open the channel (Right). Shown here are the structural superimpositions between apo-RyR2 and Ca2+ alone and between Ca2+ alone and P/Ca2+ relative to the channel domain. Red arrows indicate the direction of conformational changes. (C) The pore remains closed in the presence of 20 μM Ca2+ alone. The cytosolic view is shown for RyR2 under the 3 indicated conditions. The indicated distances were measured between the Cα atoms of the Gln4864 (open structure) or Ile4868 (closed structure) gating residue on the S6 segments in the diagonal protomers. (D) Intradomain…

Fig. 2.. FKBP12.6 stabilizes RyR2 in a closed state. (A) The EM map and atomic model of RyR2 in a complex with FKBP12.6, PCB95, and Ca2+ (F/P/Ca2+). FKBP12.6 is colored magenta. The map is shown at a contour level of 0.025. (B) FKBP12.6 serves as a stabilizer to enhance the rigidity of the HD2 and P2 domains. Shown here are the EM maps (low pass filtered to 6 Å resolution) of the P/Ca2+ and F/P/Ca2+ structures. Additional density for the HD2 and P2 domains can be resolved in the F/P/Ca2+ structure, whereas the corresponding density is absent in the P/Ca2+ structure. (C) FKBP12.6 induces the NTD and handle domains to tilt outward with respect to the central domain, likely providing a pulling force for relaxing the central domain. The NTD, handle, and central domains of the structures P/Ca2+ and F/P/Ca2+ were superimposed relative to the central domain. Red arrows indicate the direction of conformational changes. (D) The pore is kept closed in the F/P/Ca2+ structure. The indicated distances were measured between the Cα atoms of the Ile4868 gating residue on the S6 segments in the diagonal protomers.

Fig. 3.. Molecular basis for the synergistic effects of Ca2+, ATP, and caffeine. (A) Neither ATP/Ca2+ nor caffeine/Ca2+ is sufficient to open the channel in the presence of FKBP12.6. The indicated distances were measured between the Cα atoms of Ile4868 gating residue on the S6 segments in the diagonal protomers. (B) The synergistic effects of caffeine and ATP on the channel domain. The structures were superimposed relative to the CTD. (C) The binding sites of Ca2+, ATP, and caffeine on RyR2. The dotted circle represents the O-ring that is formed by the CTD, VSC, and S6C. Ca2+ is located in the cleft that is formed by the central domain and CTD. ATP is located in a pocket formed by the U-motif, CTD, and S6C. Caffeine is located at the interface formed by the U-motif and helix α4 of the central domain, CTD, and VSC. (D) Conformational changes of the central domain induced by caffeine. Shown here are the superimposed structures of F/A/Ca2+ and F/A/C/Ca2+. Arrows indicate the direction of conformational changes from the F/A/C/Ca2+ to F/A/Ca2+ structure. (E) Caffeine locks the central domain in a constitutively contracted state, and ATP strengthens the pulling force of caffeine by linking the…

Fig. 4.. Allosteric and concerted modulation of RyR2 by distinct factors. Shown here is a summary of the RyR2 structures reported in this (green box) and previous (black box) studies. Ca2+ alone induces a contraction of the central domain that provides a pulling force, facilitating the dilation of the S6 bundle. Addition of PCB95 induces further contraction of the central domain and opens the channel. FKBP12.6 induces a relaxation of the central domain, stabilizing RyR2 in a closed state. Caffeine locks the central domain in a constitutively contracted state, and ATP strengthens the effect of caffeine, stabilizing RyR2 in an open state in the presence of FKBP12.6.