Arrhythmia mutations in calmodulin can disrupt cooperativity of Ca²⁺ binding and cause misfolding

Abstract

Key points Mutations in the calmodulin protein (CaM) are associated with arrhythmia syndromes. This study focuses on understanding the structural characteristics of CaM disease mutants and their interactions with the voltage‐gated calcium channel CaV1.2. Arrhythmia mutations in CaM can lead to loss of Ca2+ binding, uncoupling of Ca2+ binding cooperativity, misfolding of the EF‐hands and altered affinity for the calcium channel. These results help us to understand how different CaM mutants have distinct effects on structure and interactions with protein targets to cause disease. Abstract Calmodulinopathies are life‐threatening arrhythmia syndromes that arise from mutations in calmodulin (CaM), a calcium sensing protein whose sequence is completely conserved across all vertebrates. These mutations have been shown to interfere with the function of cardiac ion channels, including the voltage‐gated Ca2+ channel CaV1.2 and the ryanodine receptor (RyR2), in a mutation‐specific manner. The ability of different CaM disease mutations to discriminate between these channels has been enigmatic. We present crystal structures of several C‐terminal lobe mutants and an N‐terminal lobe mutant in complex with the CaV1.2 IQ domain, in conjunction with binding assays and complementary structural biology techniques. One mutation (D130G) causes a pathological conformation, with complete separation of EF‐hands within the C‐lobe and loss of Ca2+ binding in EF‐hand 4. Another variant (Q136P) has severely reduced affinity for the IQ domain, and shows changes in the CD spectra under Ca2+‐saturating conditions when unbound to the IQ domain. Ca2+ binding to a pair of EF‐hands normally proceeds with very high cooperativity, but we found that N98S CaM can adopt different conformations with either one or two Ca2+ ions bound to the C‐lobe, possibly disrupting the cooperativity. An N‐lobe variant (N54I), which causes severe stress‐induced arrhythmia, does not show any major changes in complex with the IQ domain, providing a structural basis for why this mutant does not affect function of CaV1.2. These findings show that different CaM mutants have distinct effects on both the CaM structure and interactions with protein targets, and act via distinct pathological mechanisms to cause disease.

Allosteric Switching of Calmodulin in a Mycobacterium smegmatis porin A (MspA) Nanopore‐Trap

“…A recent study suggested that the mutant D129G lost its ability to bind Ca 2+ as a result of the separation of EF‐hands within the C‐lobe, [43] consistent with results of the MD simulations conducted in this study (Figure 3 d). To investigate this experimentally, with 0.6 μM CaM‐D129G and 4 mM Ca 2+ added to cis , nanopore measurements report a new type of event with a deeper blockage depth, similar to that of Ca‐wtCaM.…”

Binding of calcium and magnesium to human cardiac troponin C

“…It is possible that the effect of this double mutant is to reduce the binding of these cations, especially Mg 2+ through allosteric interactions. In CaM, mutation of Ca 2+ coordinating residues within the EF-hand can have structural consequences leading to altered binding kinetics (114); this is conceivable in our double mutant. Similarly, it is possible that the competition observed between Ca 2+ and Mg 2+ for binding to site II of cTnC occurs through structural perturbations, which follow binding of Mg 2+ to an allosteric site.…”

Binding of Calcium and Magnesium to Cardiac Troponin C

“…It is possible that the effect of this double mutant is to reduce the binding of these cations, especially Mg 2+ through allosteric interactions. In CaM, mutation of Ca 2+ coordinating residues within the EF-hand can have structural consequences leading to altered binding kinetics (Wang, Brohus et al 2020); this is conceivable in our double mutant. Similarily, it is possible that the competition observed between Ca 2+ and Mg 2+ for binding to site II of cTnC occurs through structural perturbations which follow binding of Mg 2+ to an allosteric site.…”