Arrhythmia mutations in calmodulin cause conformational changes that affect interactions with the cardiac voltage-gated calcium channel

Abstract

Calmodulin (CaM) represents one of the most conserved proteins among eukaryotes and is known to bind and modulate more than a 100 targets. Recently, several disease-associated mutations have been identified in theCALMgenes that are causative of severe cardiac arrhythmia syndromes. Although several mutations have been shown to affect the function of various cardiac ion channels, direct structural insights into any CaM disease mutation have been lacking. Here we report a crystallographic and NMR investigation of several disease mutant CaMs, linked to long-QT syndrome, in complex with the IQ domain of the cardiac voltage-gated calcium channel (CaV1.2). Surprisingly, two mutants (D95V, N97I) cause a major distortion of the C-terminal lobe, resulting in a pathological conformation not reported before. These structural changes result in altered interactions with the CaV1.2 IQ domain. Another mutation (N97S) reduces the affinity for Ca2+by introducing strain in EF hand 3. A fourth mutant (F141L) shows structural changes in the Ca2+-free state that increase the affinity for the IQ domain. These results thus show that different mechanisms underlie the ability of CaM disease mutations to affect Ca2+-dependent inactivation of the voltage-gated calcium channel.

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

“…As described before (Wang et al . 2018), N98S CaM displays a modest but statistically significant reduction in affinity for the IQ domain in the range of 100 nM to 10 µ m Ca 2+ . The largest effect is seen for D130G and Q136P CaM, which have decreased affinities at all Ca 2+ concentrations; in particular, the mutants have 10‐ to 1000‐fold weaker affinity for the IQ domain at 100 nM to 10 µ m Ca 2+ , with the Q136P mutation having the most severe impairment in binding.…”

“…Co‐expression and co‐purification procedures for the CaM:IQ domain complexes were described (Wang et al . 2018).…”

“…Buffered solutions, experimental methods, and curve fitting for the 5‐carboxytetramethylrhodamine (TAMRA) fluorescence anisotropy experiments were as previously described (Wang et al . 2018). Differences in the Ca V 1.2‐IQ peptide affinity between CaM mutants and WT were evaluated by a one‐way ANOVA at each Ca 2+ concentration with Dunnett's multiple comparisons test.…”

“…15 N‐labelled WT/D96V/N98S CaM was expressed and purified as previously described (Wang et al . 2018). Two hundred micromolar Ca 2+ ‐bound WT/D96V/N98S CaM was saturated with a synthetic peptide (>97% purity, Proteogenix, France) corresponding to the IQ domain of human Ca V 1.2 (DEVTVGKFYATFLIQEYFRKFKKRKEQGLVGKPS‐NH 2 ).…”

Diminished inhibition and facilitated activation of RyR2‐mediated Ca2+ release is a common defect of arrhythmogenic calmodulin mutations

“…One potential explanation for these differences may be the distinct locations of the mutations as judged by the protein crystal structures of Ca 2+ ‐bound CaM (e.g. PDB file http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2BCX, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=1CLL, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=1CFF, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2F3Y, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6GDK, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6DAF) . The F142L mutation is located at the interface between EF‐hand 3 and 4 in the CaM C‐domain, whereas the other CaM C‐domain mutations are located at Ca 2+ ‐coordinating residues in the EF‐hand loops and not part of the EF‐hands’ interface (Fig.…”