Localization of an NH2-terminal Disease-causing Mutation Hot Spot to the “Clamp” Region in the Three-dimensional Structure of the Cardiac Ryanodine Receptor

Abstract

A region between residues 414 and 466 in the cardiac ryanodine receptor (RyR2) harbors more than half of the known NH 2 -terminal mutations associated with cardiac arrhythmias and sudden death. To gain insight into the structural basis of this NH 2 -terminal mutation hot spot, we have determined its location in the three-dimensional structure of RyR2. Green fluorescent protein (GFP), used as a structural marker, was inserted into the middle of this mutation hot spot after Ser-437 in the RyR2 sequence. 2؉ release similar to that in cells expressing RyR2-wt. The three-dimensional structure of the purified RyR2 S437-GFP was reconstructed using cryo-electron microscopy and single particle image processing. Subtraction of the three-dimensional reconstructions of RyR2-wt and RyR2 S437-GFP revealed the location of the inserted GFP, and hence the NH 2 -terminal mutation hot spot, in a region between domains 5 and 9 in the clamp-shaped structure. This location is close to a previously mapped central disease-causing mutation site located in a region between domains 5 and 6. These results, together with findings from previous studies, suggest that the proposed interactions between the NH 2 -terminal and central regions of RyR2 are likely to take place between domains 5 and 6 and that the clamp-shaped structure, which shows substantial conformational differences between the closed and open states, is highly susceptible to disease-causing mutations.Cardiac arrhythmia, a leading cause of sudden death, has been linked to a number of genes encoding ion channels, including the cardiac sarcoplasmic recticulum Ca 2ϩ release channel (ryanodine receptor (RyR) type 2) 3 (1-4). To date, more than 40 mutations in RyR2 have been associated with at least two forms of cardiac arrhythmias, catecholaminergic polymorphic ventricular tachycardia (CPVT), and arrhythmogenic right ventricular dysplasia type 2 (ARVD2). Most of these mutations are located in the NH 2 -terminal region (between amino acids 164 and 466), central region (between amino acids 2246 and 2504), and the COOH-terminal region (between amino acids 3778 and 4959) of RyR2 (Fig. 1). Interestingly, those mutations in the skeletal muscle RyR (RyR1) that are linked to malignant hyperthermia (MH) and central core disease (CCD) are largely clustered in the corresponding regions of RyR1; some sites even correspond exactly (2, 4) (Fig. 1). These similar distributions suggest that the molecular mechanisms by which these disease-linked RyR1 and RyR2 mutations alter the intrinsic properties of the channel are conserved.A number of disease-linked RyR2 mutations have been expressed and functionally characterized. A common finding of these studies is that most of the RyR2 mutations enhance the activity of the channel upon stimulation (1-4). This enhancement of function activity is also a consistent feature with most of the RyR1 MH and CCD mutations (5-7). However, the precise mechanism by which channel function is altered by disease-linked mutations is not well defined. Wehrens et al. (8) ...

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

“…Our confidence in placement of the density corresponding to the N terminus of RyR1 within the cryo-EM map is substantiated by previous difference imaging with GST-RyR and GFP-RyR fusion proteins and antibody labeling ( Fig. S5) (25)(26)(27). Model placement is further validated by the match of the ␤-sheets ␤5 and ␤6 and a long helix (␣9) independently derived from the aforementioned SSEHunter results (Fig.…”

Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release

“…The first 210 amino acids of the RyR1 structure (rabbit) was at 2.5 Å resolution (PDB ID code 3HSM) (Amador 2009), and a similar fragment (amino acids 9 -205 of rabbit RyR1) was resolved at 2.9 Å resolution (PDB ID code 3ILA) (Lobo and Van Petegem 2009). The structure of the first 217 amino acids of mouse RyR2 was at 2.5 Å resolution (PDB ID code 3IM5) (Lobo and (Wang et al 2007) and was mapped to a location between subdomains 5 and 9 using green fluorescent protein insertion into the primary structure and difference mapping of cryo-EM reconstructed structures (Wang et al 2007). Notably, mutations at positions Glu161, Arg164, Arg402, and Ile404 of RyR1 and the mutations Arg169, Ile417, and Arg418 of RyR2 are located in the suggested FKBP binding pocket (Serysheva et al 2008); whereas the Ile4898Thr CCD mutation appears to be located in the proposed selectivity filter in the pore region.…”

IP3 Receptors: Toward Understanding Their Activation

“…At 30 Å resolution, the structures of the three RyR subtypes are almost indistinguishable, and because they, like the three subtypes of IP 3 R, share about 65% sequence identity, it seems reasonable to suppose that the 3D structures of all IP 3 R are also likely to be similar to each other. These studies of RyR have identified positions of critical residues within the 3D structure, the sites to which accessory proteins bind, and conformational changes associated with opening of the pore (Orlova et al 1996;Serysheva et al 2005;Wang et al 2007;Jones et al 2008). Activation of RyR is associated with considerable changes in both the pore and cytoplasmic regions: The four corners of the latter dip down toward the SR, while the central region lifts away from it (Samso et al 2009).…”

“…Such an interaction would require that the SD comes very close to the pore, but the exact location of the SD within the 3D structure of either the IP 3 R or RyR is unknown. The N-terminal of the RyR probably lies within the clamp region at the periphery of the large square cytoplasmic structure (Wang et al 2007), and it does change shape during RyR activation (Samso et al 2009). Yet, in this location the N-terminal is too far from the pore to interact directly with the TMD4-5 loop, consistent perhaps with evidence that in RyR the N-terminal may interact directly with a neighboring domain that includes residues from the central part of the primary sequence (Wang et al 2007).…”

“…The N-terminal of the RyR probably lies within the clamp region at the periphery of the large square cytoplasmic structure (Wang et al 2007), and it does change shape during RyR activation (Samso et al 2009). Yet, in this location the N-terminal is too far from the pore to interact directly with the TMD4-5 loop, consistent perhaps with evidence that in RyR the N-terminal may interact directly with a neighboring domain that includes residues from the central part of the primary sequence (Wang et al 2007). These observations and the evidence that the uncoupling peptide derived from the N-terminal of RyR2 appears to interact with residues remote from the pore (Oda et al 2005;Tateishi et al 2009), suggest that the links between the SD and pore may, at least for RyR, be indirect.…”