NMR characterization of interleukin-2 in complexes with the IL-2Rα receptor component, and with low molecular weight compounds that inhibit the IL-2/IL-Rα interaction

Nuclear magnetic resonance (NMR) methods were employed to study the interaction of the cytokine Interleukin-2 (IL-2) with the alpha-subunit of its receptor (IL-2Ralpha), and to help understand the behavior of small molecule inhibitors of this interaction. Heteronuclear H-1-N-15 HSQC experiments were used to identify the interaction surface of N-15-enriched Interleukin-2 (N-15-IL-2) in complex with human IL-2Ralpha. In these experiments, chemical shift and line width changes in the heteronuclear single-quantum coherence (HSQC) spectra upon binding of N-15-IL-2 enabled classification of NH atoms as either near to, or far from, the IL-2Ralpha interaction surface. These data were complemented by hydrogen/deuterium (H/D) exchange measurements, which illustrated enhanced protection of slowly-exchanging IL-2 NH protons near the site of interaction with IL-2Ralpha. The interaction surface defined by NMR compared well with the IL-2Ralpha binding site identified previously using mutagenesis of human and murine IL-2. Two low molecular weight inhibitors of the IL-2/IL-2Ralpha interaction were studied: one (a cyclic peptide derivative) was found to mimic a part of the cytokine and bind to IL-2Ralpha; the other (an acylphenylalanine derivative) was found to bind to IL-2. For the interaction between IL-2 and the acylphenylalanine, chemical shift perturbations of N-15 and (NH)-N-15 backbone resonances were tracked as a function of ligand concentration. The perturbation pattern observed for this complex revealed that the acylpherylalanine is a competitive inhibitor-it binds to the same site on IL-2 that interacts with EL-2Ralpha.