Subnanometer-resolution electron cryomicroscopy-based domain models for the cytoplasmic region of skeletal muscle RyR channel

Abstract

The skeletal muscle Ca(2+) release channel (RyR1), a homotetramer, regulates the release of Ca(2+) from the sarcoplasmic reticulum to initiate muscle contraction. In this work, we have delineated the RyR1 monomer boundaries in a subnanometer-resolution electron cryomicroscopy (cryo-EM) density map. In the cytoplasmic region of each RyR1 monomer, 36 alpha-helices and 7 beta-sheets can be resolved. A beta-sheet was also identified close to the membrane-spanning region that resembles the cytoplasmic pore structures of inward rectifier K(+) channels. Three structural folds, generated for amino acids 12-565 using comparative modeling and cryo-EM density fitting, localize close to regions implicated in communication with the voltage sensor in the transverse tubules. Eleven of the 15 disease-related residues for these domains are mapped to the surface of these models. Four disease-related residues are found in a basin at the interfaces of these regions, creating a pocket in which the immunophilin FKBP12 can fit. Taken together, these results provide a structural context for both channel gating and the consequences of certain malignant hyperthermia and central core disease-associated mutations in RyR1.

Fig. 1.

A 9.6-Å resolution cryo-EM density map of RyR1 in closed state. The map is displayed at the threshold level corresponding to channel molecular mass of ∼2.3 MDa and viewed from cytoplasm (A) and in a side view (B). Subregions are shown within one of the RyR1 subunits. The numbering of subregions is adopted according to a previous convention except with finer divisions (43). A subregion is interpreted as compact protein density, and the boundary of subregion tends to be weakly connected with its adjacent densities. A subregion may thus consist of a single/multiple protein fold or of a part of a protein fold. Poorly connected densities between subregions 6 and 8a and between 10 and 8b may be hinges between subregions with high local structural flexibility. The individual subregions are mapped into the distinct morphological units: clamp is formed by subregions 5, 7a, 7b, 8b, 9, and 10 from one monomer and by subregions 6 and 8a from adjacent subunit; handle is formed by subregions 3 and 4; the central rim is formed by subregions 1, 2a, and 2b; and column is formed by subregions 11 and 12.

Fig. 2.

Secondary structure elements in the structure of RyR1. (A) Two opposing RyR1 subunits from the tetramer are shown in a side view; thus, two different faces of the RyR1 subunit are seen. Subregions are numbered. Note that densities corresponding to subregion 6 are missing at the chosen high threshold. (B) Two subunits are shown as semitransparent surfaces with identified secondary structure elements. α-Helices are annotated as cylinders colored according to their locations in the map: red, TM region; green, central part of the CY region; purple, clamps; cyan, column region. β-Sheets are shown as orange surfaces. Note that a substantial fraction of the density is not assigned in this interpretation probably because of the structural flexibility or limited resolution. A dashed line indicates β-sheets in the column region that are connected with the TM region through bridging densities marked with dotted lines in A. (C) X-ray structure of KirBac1.1 channel; two subunits are removed to see the structural elements in the conduction pathway in the KirBac1.1 (11) (see also Fig. S1).

Fig. 3.

Localization of homology models for the N-terminal region of the RyR1 in the 3D map. (A) Comparative models for the N-terminal region of RyR1 and corresponding structural templates. Model 1 (residues Q12–S207) is shown with cyan ribbon; model 2 is composed of two parts, shown with yellow (residues G216–T407) and red (residues A408–Y565) ribbons. Two parts of model 2 were built and refined independently to obtain better fits to the cryo-EM map. Final models were obtained through refinement of initial models using Moulder-EM (22). Dashed lines in template structures indicate regions that were not resolved in original x-ray structures (19). (B) The RyR1 monomer is shown in a side view along with homology models docked to the clamp region. Subregions are numbered. (C) Refined homology models are shown fitted within the density region segmented from the 9.6-Å map. The segmented density region is displayed in the orientation derived from its position in B by rotation to ∼90° around the axis as shown. SSEHunter-identified α-helices and β-sheets are shown with purple cylinders and yellow solid densities, respectively. N- and C-terminal residues in both models are indicated.

Fig. 4.

Mapping of MH/CCD-linked residues onto the homology models for the N-terminal region of RyR1. Model 1 (cyan) and the model 2 (red and yellow) are shown docked within the EM density map displayed with a mesh. MH/CCD-associated residues are shown with spheres and are labeled. Labels highlighted with color mark residues exposed on the protein surface.

Fig. 5.

Identification of MH/CCD-linked residues in the FKBP12 binding pocket. The surface of the clamp in RyR1 is color-coded based on location of residues associated with MH and CCD mutations in the N-terminal homology models docked to the 3D map of RyR1. Cyan surface corresponds to residues from model 1, and yellow and red correspond to residues from model 2 (see color code used in Figs. 3 and 4). Putative surface-exposed mutation residues are labeled. The basin between subregions 3, 5, and 9 (indicated with dashed line) is proposed to form the FKBP-12 binding site in RyR1 (31, 32) and is identified to include four surface-exposed MH/CCD mutation sites (E161, R164, R402, and I404). The surface of the adjacent RyR1 subunit is shown with a mesh.