Phylogenetic and biochemical analysis of calsequestrin structure and association of its variants with cardiac disorders

Figures

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  Figure 1. Calsequestrin homologues in the vertebrate and invertebrate lineages. Empty circles indicate the gene is absent. Green circles indicate the presence of pre-duplication Casq, blue circles the presence of casq1, and red circles the presence of casq2.

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  Figure 2. Amino acid sequence alignments and calsequestrin 3D structure. (A) The 3D structure of the cardiac isoform of calsequestrin (Casq2) is shown (2VAF). The location of R33Q, L167H, K180R, D307H, P329S, G332R, and D351G Casq2 mutants are depicted in the structure. A scale of variable to conserved residues is indicated in the Figure. Mutants are shown as dot sphere. The highly conserved beta-sheet from the third thioredoxin-like fold is enlarged in the box and shown separately. The table shows the alignment of calsequestrin amino acid sequences. The degree of conservation of Casq2 sequences is color-coded using ConSurf 71. Multiple sequence alignments were inputed from the Casq1, Casq2, and Casqp alignment. Different colors represent similar/identical amino acid residues. The location of mutated residues is indicated in the table. hCASQ2, human Casq2. (B) Human Casq2 (PBD: 2VAF) with electrostatic potential surfaces. The location of the N- and C-terminus of Casq2 is indicated. Secondary structures are shown as green ribbons, the amino acid residues corresponding to the CPVT related mutations are indicated by the corresponding van der Waals surface as orange spheres. Local environments…

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  Figure 3. Calcium binding to calsequestrin mutants. (A–G) MST analysis of Ca2+ binding to mutants Casq2 (red line) and wild-type Casq2 (black line). (H) Calculated Ca2+ binding affinities of Casq2 mutants. (I) 3D structure of human Casq2 (adapted from 2VAF) with the location of mutants indicated in the Figure. The red circle depicts the location of mutations that affected Ca2 binding to Casq2.

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  Figure 4. CD analysis of Casq2 mutants. (A–G) CD analysis of Casq2 mutants (red solid line) and wild-type Casq2 (black solid line). CD analysis in the presence of 5 mM Ca2+ is indicted by black dotted lines for wild-type Casq2 and by red dotted lines for Casq2 mutants. (H) Change in % of α-helix and β-strand content with the absence or presence of Ca2+ for wild-type Casq2 and each Casq2 mutant. Negative and positive values indicate a loss or gain of secondary structure content, respectively. (I) Three-dimensional structure of human Casq2 (2VAF). Red circles depict the location of mutations with altered CD spectrum.

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  Figure 5. Limited trypsin digestion of Casq2 mutants. Wild-type Casq2 and Casq2 mutants were subjected to trypsin proteolysis in the absence (A) and presence (B) of 5 mM Ca2+ followed by SDS-PAGE. The tryptic fragments, which differed from that of wild-type Casq2 are indicated by the arrowheads. (C) The rate of proteolysis of wild-type and mutant Casq2 as a function of time of trypsin digestion. (D) The rate of proteolysis plotted as the first derivative of the fitted curve from (C).

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  Figure 6. Thermal denaturation analysis of Casq2 and Casq2 mutants. (A–G) Thermal denaturation analysis of wild-type Casq2 (black lines) or Casq2 mutants (red lines) was monitored by intrinsic tryptophan fluorescence of proteins in response to increased temperature from 35 to 95 °C. Graphs represent 3 independent measurements. (H) Inflection temperature for Casq2 and Casq2 mutants representing the temperature at which the transition from folded protein to unfolded state occurs. (I) Human Casq2 crystal structure (2VAF). Mutants with significant difference in their protein folding are indicated by red circles. The location of tryptophan residues are depicted as yellow sticks.

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  Figure 7. Ca2+-dependent polymerization of Casq2 and Casq2 mutants. (A) Coomassie blue stained SDS-PAGE of Casq2 and Casq2 mutant incubated with or without cross-linker at increasing free Ca2+ concentration. (B) Immunoblots were probed with anti-Casq2 antibodies. The full-length blots are shown in Figs. S4 and S5. (C) Quantitative analysis of Casq2 monomer (~ 50 kDa protein band) of wild-type or mutant proteins in the presence of cross-linker (from A) as a function of increased free Ca2 concentration.

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  Figure 8. Polymerization of Casq2 mutants. (A) Polymerization of Casq2 or Casq2 mutants was carried out at 167 µM free Ca2 followed by SDS-PAGE or native gel electrophoresis. Immunoblots were probed with anit-Casq2 antibodies. A representative of four independent experiments is shown. The full-length blots are shown in Fig. S6. (B) Quantitative analysis of monomeric and oligomeric forms of Casq2 mutants. (C) Human Casq2 crystal structure (2VAF). Red circles depict the location of Casq2 mutations with highly increased oligomerization.

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  Figure 9. Casq2 mutants binding to the ER luminal domain of IRE1α. (A–G) Recombinant N-terminus luminal domain of IRE1α (IRE1-NLD) protein was covalently labeled with a red fluorescent tag and incubated with increasing amounts of Casq2 or Casq2 mutant as indicated in the Figure. Normalized MST time traces are shown to the right of the graph. Each data point is the average of three independent microscale thermophoresis measurements. (H) Bar graph depicts dissociation constants for different Casq2 mutants. *p = 0.05. (I) Human Casq2 crystal structure (2VAF). The red circle on the Casq2 3D structure depicts the location of mutation with altered IRE1-NLD binding.