Fig. 5.

Small molecule binding at the AB loop quenches dynamics of mIL-2. (A) The 2D ¹H,¹³C-HMQC spectra of ILV-methyl–labeled mIL-2 in the free state (red), and with increasing concentrations of Ro 26-4550 inhibitor (1:4 molar ratio, shown in black). Data were recorded at 800 MHz, 25 °C. Fast-exchange chemical shift changes are highlighted with arrows for select methyl resonances. (B) NMR line shape analysis for the V129γ₂ and L133δ₂ methyl resonances using TITAN (Materials and Methods), with indicated equilibrium dissociation constant and errors propagated from the spectral S/N. Recorded NMR spectra are shown in blue, with simulated line shapes in red. Ratios of mIL-2 to inhibitor are indicated in each panel. (C) Two views showing the mapping of residues undergoing significant chemical shift perturbations (CSPs) onto the ribbon representation of mIL-2. Methyl groups with marked CSPs (>0.05 ppm) are shown as black spheres. The inhibitor is shown as a blue ball-and-stick diagram on the overlaid hIL-2 complex structure (PDB ID code 1M48). (D) The ¹³C single quantum CPMG relaxation dispersion profiles in the absence (purple) and presence of Ro 26-4550 at saturating concentration (black) for selected residues in the AB loop (L54δ₁), B Helix (L80δ₁), C Helix (I101δ₁), and D Helix (V130γ₂, L133δ₁, I137δ₁). CPMG experiments were performed at a ¹H field of 600 MHz and 25 °C.