Calcium‐dependent cooperativity and stability of Titin's tandem I82 ‐ I83 domains

FIGURE 1. Calpain and ankyrin repeat protein binding sites and mdm‐deletion locations within the N2A region of titin. The I‐band of titin is shown with the N2A region of titin identified between the proximal Ig segment and the PEVK region. Unique features of the tandem Ig domains in the N2A region are highlighted including two calpain‐3 binding sites, which are identified by horizontal shading. Calpain‐3 (also called p94) is a muscle‐specific calpain. One site spans the I82 and I83 domains while the other spans I80 and the insertion sequence. A cardiac ankyrin repeat protein (CARP) binding site is identified by vertical shading at the insertion sequence, abutting the I81 domain. The mdm‐deletion truncates the I83 domain and the PEVK region and is crucial for proper muscle function (Miller et al., 2003; Ono et al., 2016).

FIGURE 2. Equilibrium assays yield more cooperative unfolding for tandem I82‐I83 than for I82 + I83. Average of three trials were plotted on each graph. (a) The stability in the absence of calcium of the tandem I82‐I83 (solid black line, filled circles) yields a cooperative, two‐state unfolding event whereas the equimolar mixture of I82 + I83 (dashed line, open circles) fits a three‐state unfolding model with an intermediate at 3.5 M urea. (b) In the presence of calcium (50 μM Ca2+), the tandem I82‐I83 (solid black line, filled circles) unfolds in a cooperative, two‐state model and the equimolar mixture of I82 + I83 (dashed line, open circles) fits a three‐state unfolding model with an intermediate at 4.0 M urea. (c) The tandem I82‐I83 unfolds at higher concentrations of urea and demonstrates a more cooperative unfolding with a steeper slope for the transition in the presence of 50 μM Ca2+ (gray line) than in the absence of calcium (black line).

FIGURE 3. Superimposed 2D 15N HSQC spectra of titin domain I82 (green), I83 (red) and the tandem of I82‐I83 (blue). Selected, well resolved residues are labeled.

FIGURE 4. Strips of 3D NOESY‐HSQC spectra of the I82‐I83 tandem and I82 alone for residues G21 and V68 to indicate the changes in chemical shifts in the domain–domain interface and the additional NOE (to F121 He) that connects the two domains in the interface. Selected cross peaks from nearby resonances are indicated.

FIGURE 5. Chemical differences of equivalent residues in the individual domains I82/I83 compared to the tandem. (a) chemical shift differences for I82. (b) chemical shift differences for I83. The standard deviation of the whole set of differences is shown in red lines as integer multiples.

FIGURE 6. Solution structure family of the I82‐I83 tandem. Domain I82 is left, domain I83 is right. Separately superimposed are the top four structures from the best clusters for (a) I82OUT and (b) I82IN. (c) Structure family for I82OUT with CSP values from Figure 7 shown in color (2 * σ = yellow; 3 * σ = orange; 4 * σ = red).

FIGURE 7. The interface of domain I82 (left) and I83 (right) in the solution structure of the tandem. Heavy atoms and polar hydrogens are shown. Amino acid types are indicated in color: Green: Hydrophobic; Magenta: Hydrophilic; Red: Negatively charged; Blue: Positively charged; Orange: Glycine. Interdomain hydrogen bonds/salt bridges are indicated by lines. Selected residues are labeled.

FIGURE 8. (a) Superposition of the NMR structure of the I82‐I83 tandem onto that determined by X‐ray crystallography (both clusters of the NMR structure shown as in Figure 6, that is, I82IN in red, I82OUT in blue). I82 is on the left, I83 on the right. (b) Superposition of the crystal structure (green) on the representative NMR structure (in blue, slightly rotated compared to A to make the difference in domain orientation more evident). The principal axis for the solution structure is indicated by a magenta line to illustrate the difference in domain position between the solution and the NMR structure.

FIGURE 9. Relaxation and dynamics analysis. (a) 1H‐15N heteronuclear NOE. (b) 15N R1. (c) 15N R2. (d) Order parameter S2 for ps timescale mobility. (e) Order parameter S2 i for ns time scale mobility. (f) Exchange broadening contribution Rex to R2.

FIGURE 10. Results of NMR calcium binding experiments. (a) Chemical shift perturbations (CSP) for the addition of calcium to the I82‐I83 tandem (blue), I82 on its own (green) and I83 on its own (red) plotted against the sequence. (b) CSP values >0.02 for the tandem mapped on the structure of the tandem (red) with labels for the corresponding amino acids.