title: NMR characterization of interleukin-2 in complexes with the IL-2Ralpha receptor component, and with low molecular weight compounds that inhibit the IL-2/IL-Ralpha interaction.
abstract: 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 (1)H-(15)N HSQC experiments were used to identify the interaction surface of (15)N-enriched Interleukin-2 ((15)N-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 (15)N-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 (15)N and (15)NH 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-2Ralpha.
authors.affiliation: Structural Chemistry Group, Hoffmann-La Roche Inc., Nutley, New Jersey 07110-1199, USA.
authors.forename: S Donald
authors.initials: SD
authors.lastname: Emerson
authors.forename: Robert
authors.initials: R
authors.lastname: Palermo
authors.forename: Chao-Min
authors.initials: CM
authors.lastname: Liu
authors.forename: Jefferson W
authors.initials: JW
authors.lastname: Tilley
authors.forename: Li
authors.initials: L
authors.lastname: Chen
authors.forename: Waleed
authors.initials: W
authors.lastname: Danho
authors.forename: Vincent S
authors.initials: VS
authors.lastname: Madison
authors.forename: David N
authors.initials: DN
authors.lastname: Greeley
authors.forename: Grace
authors.initials: G
authors.lastname: Ju
authors.forename: David C
authors.initials: DC
authors.lastname: Fry
mesh_terms: D007376:Interleukin-2 / Q000378:metabolism*; D053645:Interleukin-2 Receptor alpha Subunit; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D010843:Pichia / Q000378:metabolism; D018123:Receptors, Interleukin / Q000378:metabolism*
chemical_list: C508017:IL2RA protein, human; D007376:Interleukin-2; D053645:Interleukin-2 Receptor alpha Subunit; D008024:Ligands; D018123:Receptors, Interleukin