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

• Published in: Protein science Vol. 12; no. 4; pp. 811 - 822
• Main Authors: Emerson, S. Donald, Palermo, Robert, Liu, Chao‐Min, Tilley, Jefferson W., Chen, Li, Danho, Waleed, Madison, Vincent S., Greeley, David N., Ju, Grace, Fry, David C.
• Format: Journal Article
• Language: English
• Published: Bristol Cold Spring Harbor Laboratory Press 01.04.2003
• Subjects: HSQC perturbation mapping; Interleukin‐2; Interleukin‐2 receptor; Nuclear magnetic resonance; protein binding inhibitors; protein–protein interactions
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.0232803

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