The catecholaminergic polymorphic ventricular tachycardia mutation R33Q disrupts the N-terminal structural motif that regulates reversible calsequestrin polymerization

Abstract

Calsequestrin undergoes dynamic polymerization with increasing calcium concentration by front-to-front dimerization and back-to-back packing, forming wire-shaped structures. A recent finding that point mutation R33Q leads to lethal catecholaminergic polymorphic ventricular tachycardia (CPVT) implies a crucial role for the N terminus. In this study, we demonstrate that this mutation resides in a highly conserved alternately charged residue cluster (DGKDR; cluster 1) in the N-terminal end of calsequestrin. We further show that this cluster configures itself as a ring system and that the dipolar arrangement within the cluster brings about a critical conformational flip of Lys(31)-Asp(32) essential for dimer stabilization by formation of a H-bond network. We additionally show that Ca(2+)-induced calsequestrin aggregation is nonlinear and reversible and can regain the native conformation by Ca(2+) chelation with EGTA. This study suggests that cluster 1 works as a molecular switch and governs the bidirectional transition between the CASQ2 monomer and dimer. We further demonstrate that mutations disrupting the alternating charge pattern of the cluster, including R33Q, impair Ca(2+)-CASQ2 interaction, leading to altered polymerization-depolymerization dynamics. This study provides new mechanistic insight into the functional effects of the R33Q mutation and its potential role in CPVT.

FIGURE 1.

The N terminus of CASQ is highly conserved and involved in protein folding. A, multiple sequence alignment of the N terminus. The number of amino acids before the mature protein is indicated to the left of each sequence. Identical residues in the alignment are indicated by white letters in red boxes, similar residues are shown as gray letters in cyan boxes, and a blue frame indicates similarity across groups. The alternately charged residue clusters 1 (DGKDR) and 2 (EKNLK) are labeled. The CPVT-related residue Arg³³′ is located in cluster 1. The accession numbers for each sequence and the complete alignment are provided in the legend to supplemental Fig. S1. Gaps in alignment are represented as dots. Alignment was generated using the program ESPRIPT (42). Violet triangles at the bottom indicate the positions of deletion mutations: Δ5CASQ2 (Δ5), Δ10CASQ2 (Δ10), and Δ20CASQ2 (Δ20). B, superposition of the monomer and dimer forms of CASQ2. One of the major structural differences can be seen in the N terminus. The loop formed by cluster 1 (DGKDR) is highlighted by a black rectangle. C, far-UV CD spectra of different N-terminal deletion mutants. Deletion mutants Δ10CASQ2 and Δ20CASQ2 failed to fold, but mutant Δ5CASQ2 could fold to a conformation equivalent to that of the WT protein. mdeg, millidegrees.

FIGURE 2.

Mutations in cluster 1 affect CASQ stability and polymerization. A, comparison of secondary structural content in cluster 1 mutants. The cluster 1 mutants lose secondary structural content rapidly at high temperatures. (The actual CD spectra are shown in supplemental Fig. S3.) B and C, CD spectra of cluster 1 and 2 mutants, respectively, in the presence of 5 mm CaCl₂. The CD spectra of R33Q, K31A-R33A, and D29A-D32A show highly reduced ellipticity, suggesting random aggregation. The K40A-K43A mutation had an intermediate effect, but the E39A mutation had no effect, and its CD spectrum is similar to that of the WT protein. D, polymerization and depolymerization of the WT protein and R33Q. The WT protein and mutant were treated with Ca²⁺ and chelated with EGTA as described under “Experimental Procedures.” WT CASQ regained the native conformation upon Ca²⁺ chelation by EGTA (∼3 mm), whereas R33Q failed to regain the native conformation, indicating that reversibility of CASQ2 polymerization is affected by the R33Q mutation. mdeg, millidegrees.

FIGURE 3.

Conformational changes in cluster 1 mutants as analyzed by turbidimetric assay. Aggregation measured at 350 nm (A) and 600 nm (B) showed that cluster 1 mutants R33Q, K31A-R33A, and D29A-D32A are Ca²⁺-insensitive at low calcium concentrations. Shown is the percentage of protein precipitated due to Ca²⁺-induced aggregation (C) and Ca²⁺ chelation mediated by EGTA (D) as described under “Experimental Procedures.” Cluster 1 mutants required higher EGTA concentrations to become resolubilized. For EGTA-mediated Ca²⁺ chelation, the WT protein and cluster 1 mutants were first aggregated with 8 mm CaCl₂. The replot of aggregation-disaggregation of each of the cluster 1 mutants is shown in supplemental Fig. S5.

FIGURE 4.

Cluster 1 mutants have impaired Ca²⁺ response. The trypsin digestion pattern of cluster 1 mutants is different from that of the WT protein. A, lanes 1–6, untreated WT protein and incubation with 0, 1, 2, 5, and 10 mm CaCl₂, respectively; lanes 7–12, untreated R33Q mutant protein and treatment with 0, 1, 2, 5, and 10 mm CaCl₂, respectively. B, lanes 1–6, untreated D29A-D32A mutant protein and treatment with 0, 1, 2, 5, and 10 mm CaCl₂, respectively; lanes 7–12, untreated K31A-R33A mutant protein and treatment with 0, 1, 2, 5, and 10 mm CaCl₂, respectively. The M lanes are protein markers (Bio-Rad). The differences in the tryptic fragments are highlighted with black arrows.

FIGURE 5.

Organization of cluster 1 into a ring system to function as a molecular switch. The dipoles emanating from alternately charged residues in the ring system are arranged such that they can support the coordinated conformational flip of Lys³¹-Asp³², resulting in a H-bond network. Black dashes represent H-bonds. A, location of cluster 1 in the dimer. The cluster located at the interface of the dimer in the form of a ring system regulates formation of a H-bond network to stabilize dimer conformation. The intermolecular space (shown in pink) is the site of helix-loop and helix-helix interactions shown in detail in Fig. 6A. B, conformational flip of Asp³². Superimposition studies of the monomeric (red; endo-conformer) and dimeric (green; exo-conformer) structures suggested the conformational flipping of Asp³². Asp³² and Lys⁶⁴ (of chain B, shown in orange) of the dimer are shown in ball and stick form, and Asp³² of the monomer is shown in tube form. C and D, comparison of the ring system in the dimer and monomer, respectively. The ring system in the monomer has Lys³¹ exposed outward and Asp³² oriented inward. Arg³³ regulates the outward flip of Asp³² to interact with Lys⁶⁴ and Lys⁶⁸ of chain B. The flip of alternately charged residues Lys³¹ and Asp³² suggests its plausible role in Ca²⁺ sensing and polymerization.

FIGURE 6.

Stabilization of the CASQ dimer by the formation of a H-bond network. A, intermolecular surface of the dimer. The structure of the front-to-front dimer between chains A and B is stabilized through intermolecular H-bonding by loop-helix interactions of Asp³² with Lys⁶⁸ and Lys⁶⁴ (red ellipse) and helix-helix interactions of Lys⁶⁸ with Glu⁷⁴ and Glu⁷⁸ (black triangle). The surface of chain A is shown in pink, and that of chain B is shown in blue. The interacting surface has overlapping pink and blue colors. B, the H-bond network formed by the DGKDR ring system in a dimer. Black dashes represent H-bonds. The conformational flip of Lys³¹-Asp³² brings together helices from chains A (in red) and B (in green) into proximity and is stabilized by the H-bond network.