Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias

X-ray and solution structures of the human RyR2 N-terminal region were obtained under near-physiological conditions. The structure exhibits a unique network of interactions between its three domains, revealing an important stabilizing role of the central helix.

. SAXS data were measured at four different concentrations (Table S4). The scattering curve of the particle was obtained by averaging eight measurements and subtracting average buffer scattering from sample scattering. There was a dependence of R g on sample concentration (Table S4) indicating inter-particle interactions, and therefore R g values were extrapolated to infinite dilution.
The difference between R g values calculated from Guinier approximation and from the P(r) function might indicate a slight heterogeneity of the sample, which correlated with the 31.2% polydispersity of the monomeric peak (Borko et al., 2013). Adequate protein folding was indicated by the Kratky plot.
The Porod volume of the particle was ≈152 000 Å 3 , which gave a molecular weight of ≈76 kDa. The difference of 8 kDa from the expected hRyR2 1-606 MW of 67.8 kDa likely indicates shape heterogeneity caused by flexible regions of the protein. The position of the maximum of the pair distribution function at a short distance and the extended tail of the function indicate that some segments of the structure might be extended.
The solution structure of hRyR2 1-606 was determined by ab initio modeling with GASBOR using simulated annealing. All created models had a similar shape. The model with χ 2 and NSD values of 0.95 and 1.538, respectively, was chosen for further analysis.

S1.1. Determination of relative domain orientation
Previous measurements of relative domain orientation in the IP3R1 were performed relative to the long α-helix in the suppressor domain (equivalent to domain A of the RyR), which is not present in RyR1 and not resolved in RyR2. To obtain an unequivocal representation of the domain positions, individual domains were represented by the CA-atoms of residues present in all published structures and in 4JKQ, and their position and shape were characterized by their inertia ellipsoids in CHIMERA.
( Figure S4). To determine, which of the characteristics of inertia ellipsoids were significantly changed in IP3R1 upon binding of inositol trisphosphate, a similar calculation was performed for the   equivalent domains of all chains of the known IP3R1 N-terminal structures (3T8S, 4UJ0, 4UJ4; three Ins 3 P-bound and three Ins 3 P-free structures). It was assumed that the ligand-free structure represents the closed channels and the ligand-bound structure represents the open channel. To estimate the changes occurring in RyR channels, the A, B, C domains of 4JKQ independently docked into cryoEM maps of the RyR1 in the closed and open state (Samso et al., 2009) were analyzed in the same manner. S1.2. Docking the crystal structure and the model into cryo-electron microscopy maps.
The crystal structure as well as the model could be docked into electron density maps of the RyR1 channel (EMD 1275(EMD , 1606(EMD , 1607(EMD , 1274. The ADP_EM and SITUS software packages provided essentially identical positions, similar to those previously reported for the 2XOA structure of oRyR1 1-559 (Tung et al., 2010). Docking contrasts were not significantly different between ADP_EM and SITUS. Since the RMSD between the symmetrical copies of the first four best fits was much smaller in SITUS (0.063 ± 0.020 and 0.069 ± 0.021 Å for crystal structure and model, respectively) than in ADP_EM (4.11 ± 0.25 and 3.43 ± 0.28 Å for crystal structure and model, respectively), SITUS results were analyzed further. The cross-correlation coefficients of the second-best symmetrically independent fit were 0.52 ± 0.04 of the best fits for both, crystal structure and model, indicating a

S1.3. CD spectroscopy
Solution secondary structure and thermal stability of hRyR2 1-606 wild type and mutant proteins were assessed by far-UV CD spectroscopy using 1-nm bandwidth, 0.2-nm steps, 3 to 5 s per point accumulation time. Prior to measurements, samples were centrifuged (1h, 14 000 g, 4°C). The buffer was the same as used for crystallization of the wild type protein except that NaCl was replaced by NaF for increased transparency. Spectra were recorded from 260 to ≤ 190nm at 4°C, and from 20°C on in 5°C intervals up to precipitation indicated by a steep rise in the dynode voltage. Protein concentration was determined from the absorbance at 280 nm assuming extinction coefficients as calculated from the amino acid composition (Pace et al., 1995).

S1.4. Information about macromolecular production, crystallization and data collection and processing.
Production, crystallization and data collection have been described previously (Borko et al., 2013). Table S5, S6 and S7.

S2. Multimedia Files
Supplementary Movie S1 Docking of the 4JKQ structure and its domains A, B, C into RyR cryo-EM maps. Top view (from the cytoplasm into SR lumen) and side view of pseudo-atomic models of the 4JKQ structure of hRyR2 1-606 (domains A, B, C shown in blue, green and red, respectively)     *Rg from the Guinier approximation, **Rg from the P(r) function, ***extrapolation to zero concentration cconcentration, calculated from the absorbance at 280nm and A0.1%,280nm, 1cm = 1.1 mg/ml Rgradius of gyration I(0)scattering intensity at 0° Guinier pointslinearity region for Rg estimation, number of Guinier points in parenthesis.
Dmaxmaximum dimension of the particle Vvolume of the particle

Figure S5
The cavity among the symmetry related molecules in the crystal. The molecules, in which the last visible residue (Asn544, red spheres) is facing the cavity, are shown in red, while the molecules, in which Asn544 is facing to cavities above and below the depicted region, are shown in blue.