Fig. 3.

Effects of a conserved helical capping motif on IL-2 dynamics and function. (A) Crystal structure of mIL-2 (schematic) bound to JES6-1 (blue surface) with a detailed view of the mIL-2/JES6-1 interface on the AB loop and B helix of the cytokine (PDB ID 4YQX). C-capping hydrogen bonds involving the R52 side chain are shown as black lines. (B) The AB loop in mIL-2 adopts either an uncapped “closed” conformation (apo state) or a capped “open” conformation (JES6-1–bound state). The open conformation is stabilized by an intramolecular C-capping interaction between the side chain of R52 and the backbone of M42-Y45-R46. Black dashes indicate hydrogen bonds. The R52 side chain does not contribute to intramolecular stabilizing interactions in the closed conformation. Black dashed arrow shows the direction of loop movement between the closed and open states. (C) Close-up view of the AB loop in the closed and open conformations. The R52A mutation eliminates the C-capping interactions. (D) Sequence conservation pattern in the AB loop region in various IL-2 orthologs. (E) Comparison of ¹³C-CPMG relaxation dispersion profiles for L80δ₂, I101δ₁, and L133δ₁ of WT mIL-2 (purple) and R52A (green), at 600 MHz and (F) ¹³C-CEST profiles for WT mIL-2 (red) and R52A (green). Conformational exchange profiles throughput the mIL-2 structure are quenched by the R52A mutation. Experimental errors in CPMG and CEST data are determined as described in Fig. 1. (G) Thermodynamic fingerprints of the interactions between WT and R52A mIL-2 with JES6-1. Isothermal titration calorimetry thermographs and curve fits for titrations. The R52A mutation leads to a three-order-of-magnitude reduction in binding affinity. To minimize enthalpy of solvation effects, all experiments were performed in 20 mM phosphate buffer, pH 7.2, 150 mM NaCl. Data shown are representative of duplicate experiments. (H) STAT5 phosphorylation (pSTAT5) response to WT and R52A mIL-2 in the free state or JES6-1 immunocomplexes treatment in IL-2Rα⁺ mouse CTLL-2 cells. R52A shows attenuated immunomodulation by the JES6-1 Ab while signaling via IL-2Rα⁺ is at similar levels to WT. μcal, microcalorie; kcal, kilocalorie; MFI, median fluorescence intensity.