Arrhythmogenic Calmodulin Mutations Can Disrupt the Globular Structure and Uncouple Ca2+ Binding Cooperativity

Abstract

Calmodulin (CaM) is a highly conserved Ca2+ sensor that modulates hundreds of proteins including the L-type voltage-gated Ca2+ channel CaV1.2 and cardiac Ryanodine Receptor (RyR2). Mutations in CaM are associated with life-threatening arrhythmia syndromes such as early-onset long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). In the heart, CaM interacts with the C-terminal IQ domain of CaV1.2 to facilitate calcium dependent inactivation (CDI), and several CaM disease mutants were found to suppress CaV1.2 CDI and have decreased Ca2+ affinity. Here, we utilize structural biology techniques and binding assays to characterize arrhythmia-linked CaM variants and their interactions with the CaV1.2 IQ domain. From the crystal structure, we find one LQTS-associated variant (D129G) causes unfolding of the C-lobe that completely disrupts the globular structure and a loss of Ca2+ binding in EF-hand 4. Another mutation (Q135P) severely reduces CaM's affinity for the IQ domain, and shows changes in the CD spectra compared to wild-type Ca2+/CaM when unbound to the IQ domain. The N97S CaM variant displays uncoupled Ca2+ binding cooperativity between EF-hands 3 and 4, resulting in a C-lobe that can have either one or two Ca2+ ions bound. An N-lobe variant (N53I) linked to CPVT does not exhibit significant differences in complex with the IQ domain, in agreement with past studies which show N53I CaM does not affect CaV1.2 regulation. These results confirm that different CaM variants have distinct effects on ion channel regulation and act via diverse molecular mechanisms to cause disease.