The Ryanodine Receptor N-Terminal Disease Hot Spot Intersubunit Interface is Disrupted by Channel Opening and Affected by Disease Mutations Acting via Long-Range Structural Changes

Abstract

Ryanodine Receptors (RyRs) are intracellular calcium-release channels acting as one of the key regulatory elements in the excitation-contraction coupling. More than 350 mutations have been found in RyRs that are known to underlie severe genetic diseases. Mutations in the skeletal muscle isoform (RyR1) are associated with malignant hyperthermia (MH) and central core disease (CCD), while mutations in the cardiac isoform (RyR2) cause catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia (ARVD). Most mutations confer a gain of function, but the precise mechanisms that explain enhanced channel opening up to the molecular scale have remained elusive. Here we present pseudo-atomic models of the N-terminal disease hot spot in the open and closed states of the RyR, along with crystal structures of several disease mutants. The data show that the intersubunit interfaces formed by tetrameric N-terminal disease hot spots are disrupted upon channel opening in wild-type RyRs. This intersubunit interface harbors 19 disease mutations, the largest cluster within the N-terminal region, indicating the vulnerability of this interface in channel regulation. We present crystal structures and thermal stabilities of nine disease mutants located at other interfaces. The effect of most mutations are destabilizing to the protein, with decreases in melting temperatures as large as ∼10°C. Buried disease mutations cause structural changes to the intersubunit interface, while mutations affecting ionic pairing at the intra-subunit interface significantly alter relative domain orientations. Mutations far away from the intersubunit interface can thus affect these contacts via long-range conformational changes. These results illuminate the intersubunit interface between N-terminal disease hot spots as a prime target for disease mutations through direct or indirect conformational changes.