The Deletion of Exon 3 in the Cardiac Ryanodine Receptor Is Rescued by β Strand Switching

Abstract

Mutations in the cardiac Ryanodine Receptor (RYR2) are linked to triggered arrhythmias. Removal of exon 3 results in a severe form of catecholaminergic polymorphic ventricular tachycardia (CPVT). This exon encodes secondary structure elements that are crucial for folding of the N-terminal domain (NTD), raising the question of why the deletion is neither lethal nor confers a loss of function. We determined the 2.3 Å crystal structure of the NTD lacking exon 3. The removal causes a structural rescue whereby a flexible loop inserts itself into the β trefoil domain and increases thermal stability. The exon 3 deletion is not tolerated in the corresponding RYR1 domain. The rescue shows a novel mechanism by which RYR2 channels can adjust their Ca²⁺ release properties through altering the structure of the NTD. Despite the rescue, the deletion affects interfaces with other RYR2 domains. We propose that relative movement of the NTD is allosterically coupled to the pore region.

Roles of the NH2-terminal Domains of Cardiac Ryanodine Receptor in Ca2+ Release Activation and Termination

“…The intra-and intersubunit interactions between domains A and B are believed to be important for stabilizing the closed state of the channel. Disease mutations located in interfaces between domains A and B would weaken these interactions, thus facilitating channel opening (7)(8)(9)(10)(11)(12)(13)(14). Del-A would be expected to remove both intra-and intersubunit interactions between domains A and B, leading to destabilization of the closed state and channel activation.…”

“…The recently solved crystal structures of the NH 2 -terminal region of RyR have provided novel insights into the structural basis of disease mechanisms associated with the NH 2 -terminal mutations (7)(8)(9)(10)(11)(12)(13)(14). The three-dimensional structure of the NH 2 -terminal region of RyR contains three domains: domain A (residues 1-217), domain B (residues 218 -409), and domain C (residues 410 -543) (9).…”

Crystal structures of wild type and disease mutant forms of the ryanodine receptor SPRY2 domain

“…The cap regulates the ability of the channel to open or close, turning RyRs into enormous allosteric membrane proteins 11 . A number of crystal structures have been reported, focusing mostly on the amino-terminal (N-terminal) three domains [12][13][14][15][16][17][18] , and a single domain containing a phosphorylation hot spot loop 19,20 .…”

Themes and Variations in ER/SR Calcium Release Channels: Structure and Function

“…In addition to point substitutions within ABC, deletion of exon 3 encoding a portion of domain A in RyR2 was linked to CPVT and other heart abnormalities in 16 members from two separate families (8,79). Remarkably, the structural integrity of RyR2-A is rescued via the insertion of a flexible loop into the ␤-trefoil domain affected by the deletion resulting in enhanced thermal stability (68). Hence, CPVT is likely manifested by a disruption of interfaces with other domains for individuals carrying this exon 3 deletion (68).…”

“…Remarkably, the structural integrity of RyR2-A is rescued via the insertion of a flexible loop into the ␤-trefoil domain affected by the deletion resulting in enhanced thermal stability (68). Hence, CPVT is likely manifested by a disruption of interfaces with other domains for individuals carrying this exon 3 deletion (68). The distribution of mutations on interand intra-molecular interfaces suggests that gating of the channel is allosterically coupled to the movement of individual domains relative to each other and on different subunits.…”