An Alternately Charged Residue Cluster at the N-terminal End Forms a Ring System and Dynamically Regulates Calsequestrin Polymerization

Abstract

Calsequestrin (CASQ2) mediated calcium buffering and release is the key for muscular contraction and relaxation. Upon Ca2+ binding CASQ2 undergoes polymerization in a linear fashion by front-to-front dimerization and back-to-back packing to form the wire-shaped structures as observed by electron microscopy. Being enriched in negatively charged residues, the C-terminal half of the molecule has been considered to be important for Ca2+ binding capacity and function of CASQ. However, the recent finding of R33Q mutation leading to lethal Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) in human indicates importance of N-terminal end. By protein sequence analysis we have found a cluster (DGKDR) of alternating positively and negatively charged residues in the N-terminal end, that include residue R33, conserved from C. elegans to both CASQ isoforms in human. Systematic deletion and charge neutralization mutagenesis was coupled to circular dichroism, limited proteolysis, turbidimetric assays and computational molecular dynamics to illustrate that the cluster works as a molecular switch. Molecular dynamics studies illustrate the dipolar arrangement in the cluster brings about a critical flip of D32 residue essential for stabilization of dimer by formation of hydrogen bond network and arrange the cluster into a ring system. Results show that Ca2+-induced CASQ2 aggregation is reversible, non-linear and can be resolubilized to native conformation by Ca2+-chelation with EGTA. However CASQ2 mutation with alteration in the charge pattern in the cluster, including the R33Q mutation, disrupt the ring system and reduce the backbone flexibility, thus impairing the response to Ca2+-induced aggregation and Ca2+-chelation by EGTA leading to loss of reversibility of polymerization. We propose that under increased physiological demands the R33Q mutant fails to undergo dynamic conformational interconversions necessary to cope with increased Ca2+ handling and thereby lead to CPVT.