Fig. 2. RCAN1_core domain, amino acids 128 to 164, folds-upon-binding CN.

(A) Secondary-structure propensity data plotted against RCAN1_core residue numbers (SSP > 0, α helix; SSP < 0, β strand). RCAN1_core, blue; CNA-bound RCAN1_core, pink. (B) Overlay of the 2D [¹H,¹⁵N] TROSY spectrum of free ¹⁵N-labeled RCAN1_core (blue) with the 2D [¹H,¹⁵N] TROSY spectrum of (²H,¹⁵N)-labeled RCAN1_core bound to (²H)-labeled CNA (pink). Arrows indicate the peak shifts in RCAN1_core upon binding CN. (C) 2mF_o − DF_c electron density map (blue) contoured at 1σ corresponding to the RCAN1_core PxIxIT motif (orange sticks; PxIxIT motif residues underlined) bound to CN (gray). CN residues that form the PxIxIT motif hydrophobic docking pocket shown as sticks and labeled. (D) Structure of RCAN1_core:CNA complex obtained using NMR and x-ray data corefinement. RCAN1_core, orange (PxIxIT motif shown as sticks; strand β1, arrow, helix α1, cylinder); CNA, white surface with the CNA residues that experience CSPs upon RCAN1_core binding shown in blue (>2σ SD) or light blue (>1σ SD); dark gray surface corresponds to unassigned residues. Residues that experience CSPs are also labeled. (E) Overlay of the five lowest-energy corefined structures. (F) Overlay of the 2D [¹H,¹⁵N] TROSY spectra of (²H,¹⁵N)-labeled CNA in the absence (black) and presence (red) of the RCAN1_core. Peaks that shift in the presence of the RCAN1_core are shown with arrows and labels. Label colors correspond to chemical shift perturbation deviation magnitudes [see (D)].