Localization of a disease-associated mutation site in the three-dimensional structure of the cardiac muscle ryanodine receptor

Abstract

The cardiac muscle ryanodine receptor (RyR2) functions as a calcium release channel in the heart. Up to 40 mutations in RyR2 have been linked to genetic forms of sudden cardiac death. These mutations are largely clustered in three regions of the sequence of the polypeptide: one near the N terminus, one in the central region, and the third in the C-terminal region. The central region includes 11 mutations, and an isoleucine-proline motif (positions 2427 and 2428) in the same region is predicted to contribute to the binding of FKBP12.6 protein. We have mapped the central mutation site in the three-dimensional structure of RyR2 by green fluorescent protein insertion, cryoelectron microscopy, and single-particle image processing. The central mutation site was mapped to a "bridge" of density that connects cytoplasmic domains 5 and 6, which have been suggested to undergo conformational changes during channel gating. Moreover, the location of this central mutation site is not close to that of the FKBP12.6-binding site mapped previously by cryoelectron microscopy.

FIGURE 1. The 40 mutations in RyR2 sequence implicated in sudden cardiac death

These mutations are largely clustered in three regions of the sequence (gray boxes). The ~500 amino acids (AA) beginning at the C terminus compose the transmembrane domain (TM; yellow box). The sequences of the three RyR isoforms differ primarily in the three divergent regions (DR; pink boxes). The proposed Ca²⁺ sensor, the calmodulin-binding site, a phosphorylation site, the proposed pore-forming segment, and a predicted FKBP12.6-binding site are indicated. GFP flanked by two glycine-rich spacers was inserted in the middle of the central mutation region after Ser-2367 (green oval).

FIGURE 2. Biochemical characterization of RyR2_S2367-GFP

a, Ca²⁺ release activity detected following the addition of the RyR2 activator caffeine (Caff; C′ = 0.25 mm, C = 2.5 mm) or ryanodine (Ryd; R = 50 μM) to RyR2_S2367-GFP cDNA-transfected HEK293 cells as measured by fluo-3 acetoxymethyl ester fluorescence intensity; b, [³H]ryanodine binding to the purified RyR2_S2367-GFP proteins at various Ca ²⁺ concentrations; c, SDS-PAGE and Western blot of purified RyR2_wt (lanes 1) and RyR2_S2367-GFP (lanes 2). CBB, Coomassie Brilliant Blue.

FIGURE 3. Cryo-EM of RyR2_S2367-GFP

A portion of a cryoelectron micrograph of the purified RyR2_S2367-GFP proteins embedded in a thin layer of vitreous ice is shown. The tetrameric structure of RyR2_S2367-GFP is well preserved. Several individual RyR2_S2367-GFP particles are circled. Scale bar = 600 Å.

FIGURE 4. Two-dimensional averages of RyR2_wt and RyR2_S2367-GFP

a, two-dimensional average of RyR2_S2367-GFP (n = 251 particle images) in the “bottom” view. b, two-dimensional average of RyR2_wt (n = 269 particle images) in the same view. c, difference map obtained by subtraction of b from a. The “top” and bottom views of RyR2 (both 4-fold symmetric) are the most frequent orientations assumed by RyR2 particles on EM grids; the bottom view depicted here represents the projection of the receptor as seen from the lumen side of the sarcoplasmic reticulum. The largest positive differences shown in c, corresponding to the additional masses due to the GFP insertions, are seen as bright white areas in the clamp domains (circled). d, map of statistically significant regions of difference obtained by the t test displayed at a >99.9% confidence level. The width of each frame is 544 Å.

FIGURE 5. Three-dimensional localization of GFP inserted at Ser-2367

a, a surface representation of the three-dimensional reconstruction of RyR2_S2367-GFP is shown in green. b, a difference map (RyR2_S2367-GFP − RyR2_wt) shown in green is superimposed on the three-dimensional reconstruction of RyR2_wt (blue). Each of the green regions of the difference map corresponds to a GFP insertion in the central mutation region of a RyR2 subunit. The three-dimensional reconstructions in both panels are shown in three views: left, top view from the cytoplasmic surface that in situ would face the transverse tubule membrane; middle, bottom view that would face the sarcoplasmic reticulum lumen; right, side view. The side view shows that RyR2 has a small transmembrane assembly (TA) and a large cytoplasmic assembly as the main mass of the protein. The numerals 1–10 on the cytoplasmic assembly indicate the distinguishable domains according to previous nomenclature (26).

FIGURE 6. Relationship of Ser-2367 to other landmarks on the RyR three-dimensional architecture

a, Ser-2367 is not close to the FKBP12.6-binding site in the three-dimensional structure of RyR. The binding sites of FKBP12 and FKBP12.6 have been mapped to the same location on the three-dimensional structures of RyR1 and RyR2 (magenta dots) (32). b, Ser-2367 lies adjacent to the N terminus and the N-terminal region mutation hot spot. The GST tag fused to the N terminus of RyR3 was mapped to the center of the clamp region (red) (23), and a region containing much of the N-terminal mutation region was mapped to domain 5 (yellow in one clamp structure) according to the work of Serysheva et al. (41). c, Ser-2367 is located in the clamp region, which is proposed to contact the DHPR. DHPR tetrad arrays are shown as semitransparent dark rings, which are superimposed on the RyR1 ordered arrays (from Paolini et al. (50)); the centers of the tetrads are shown in yellow. Green, red, and cyan dots represent the central mutation region and divergent regions 2 and 3, respectively.