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Protein Science (2003), 12:811-822.
Copyright © 2003 The Protein Society

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

S. Donald Emerson1, Robert Palermo2, Chao-Min Liu, Jefferson W. Tilley, Li Chen, Waleed Danho, Vincent S. Madison2, David N. Greeley, Grace Ju and David C. Fry

Structural Chemistry Group, Hoffmann-La Roche Inc., Nutley, New Jersey 07110-1199, USA

Reprint requests to: David C. Fry, Hoffmann-La Roche Inc., Bldg. 123, Room 3115, 340 Kingsland Street, Nutley, NJ 07110-1199, USA; e-mail: david.fry{at}roche.com; fax: (973) 235-6084.

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-2R{alpha}), 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-2R{alpha}. 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-2R{alpha} 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-2R{alpha}. The interaction surface defined by NMR compared well with the IL-2R{alpha} binding site identified previously using mutagenesis of human and murine IL-2. Two low molecular weight inhibitors of the IL-2/IL-2R{alpha} interaction were studied: one (a cyclic peptide derivative) was found to mimic a part of the cytokine and bind to IL-2R{alpha}; 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-2R{alpha}.

Keywords: Nuclear magnetic resonance; Interleukin-2; Interleukin-2 receptor; protein–protein interactions; HSQC perturbation mapping; protein binding inhibitors


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