Figure 1.
Structure of the CBD12 tandem.
(A) Crystal structure of CBD12-E454K in cartoon representation. CBD1 and CBD2 are colored orange and red, respectively. The rectangles frame a zoom perspective as depicted in panels B (blue), C (magenta) and D (green). Green and blue spheres depict Ca2+ ions and water molecules, respectively. Dotted black lines denote electron density chain breaks in the protein. (B–D) Residues with buried surfaces in the interface are depicted as sticks, with their electron density contoured at 1.5 σ (blue mesh).
Table 1.
Crystallographic statistics.
Figure 2.
Conservation analysis of CBD domains.
(A) CBD12-E454K structure colored according to the conservation score of each residue. (B) Conservation score for each residue. Negative values indicate conservation while positive values indicate variability. The sequence refers to the cardiac splice-variant, which is 35 residues longer than the brain spliced variant used in our study. (C) Conservation of interface residues located on the G-strand of CBD1 and BC-loop of CBD2.
Figure 3.
Ca2+ coordination in the CBD12-E454K crystal structure (orange) and in the CBD1-WT crystal structure (cyan, PDB 2DPK). Residues coordinating Ca2+ are depicted as sticks.
Figure 4.
Stopped flow analysis of CBD12-E454K and CBD12-R532A
. (A) Monophasic (uncoupled CBDs) and biphasic (coupled CBDs) dissociation kinetics of two Ca2+ ions from the Ca3-Ca4 sites, measured by stopped-flow techniques. Occupied sites are denoted by filled circles, whereas open circles represent empty sites. (B) Representative traces of Ca2+ dissociation kinetics from CBD12-WT, CBD12-E454K and CBD12-R532A. Ca2+ dissociation kinetics of CBD12-WT were fit to a double exponential curve with kf = 5.3±0.04 s−1 and ks = 0.57±0.001 s−1. The trace of CBD12-E454K was fit to a double exponential curve with kf = 52.2±1.04 s−1 and ks = 0.73±0.001 s−1. The representative trace of CBD12-R532A was fit to a single exponential curve with kf = 3.6±0.01 s−1. (C) Bars represent the mean ± S.E values of the “fast” phase (kf) of Ca2+ dissociation (n = 6) and the mean ± S.E of slow off-rates (ks) (n = 6). For CBD12-R532A, in which monophasic dissociation is observed, kf and ks are identical.
Figure 5.
SAXS analysis of CBD12 proteins
Bead model reconstruction was performed for each protein on the basis of experimental SAXS measurements. The experiments were done in the absence (left column) and presence (right column) of Ca2+, for the wild-type CBD12-WT (A), CBD12-7A (B) CBD12-R532A (C) and CBD12-E454K (D). The CBD1 domain (orange, PDB code: 2FWS) and the CBD2 domain (red, PDB code: 2FWU) are shown as cartoon and surface, and manually fit to the bead model shape (blue mesh), excepting panel A and D, right, where the cartoon is a depiction of the current crystal structure.
Table 2.
Dmax and ks values of CBD12 mutants.
Figure 6.
Superposition of CBD12 from NCX and CALX.
The structures of CBD12-E454K from NCX1 and of CBD12-1.1 and CBD12-1.2 from CALX (PDB codes 3RB5 and 3RB7, respectively) are colored cyan, black and green, respectively. The indicated values represent the hinge angle between CBD1 and 2 as defined by Cα atoms K373, H501 and E647 (NCX CBD12-E454K) and R443, H553 and I692 (CALX CBD12-1.1 and CBD12-1.2).