Localization in human interleukin 2 of the binding site to the alpha chain (p55) of the interleukin 2 receptor.

Abstract

Localization in human interleukin 2 of the binding site to the a chain (p55) ABSTRACTHuman interleukin 2 (IL-2) analogs with defined amino acid substitutions were used to identify specific residues that interact with the 55-kDa subunit (p55) or a chain ofthe human IL-2 receptor. Analog proteins containing specific substitutions for Lys-35, Arg-38, or Lys-43 were inactive in competitive binding assays for p55. All of these analogs retained substantial competitive binding to the intermediate-affinitj p70 subunit (J3 chain) of the receptor complex.The analogs varied in ability to interact with the high-affinity p55/p70 receptor. Despite the lack of binding to p55, all analogs exhibited significant biological activity, as assayed on the murine CTLL cell line. The dissociation constants ofArg-38 and Phe-42 analogs for p70 were consistent with intermediateaffinity binding; theKd values were not significantly affected by the presence of p55 in binding to the high-affinity IL-2 receptor complex. These results confirm the importance of the B a-helix in IL-2 as the locus for p55-receptor binding and support a revised model of IL-2-IL-2 receptor interaction.Human interleukin 2 (huIL-2) is a potent immunoregulatory cytokine, the biological effects ofwhich are mediated through binding to specific receptors on the surface of target cells. These interleukin 2 (IL-2) receptors (IL-2Rs) are present in at least three forms that bind IL-2 with high (Kd = 10-50 pM), intermediate (Kd = 0.8-2 nM), or low (Kd = 10-30 nM) affinity (1). The high-affinity IL-2R complex consists of at least two receptor subunits, the a chain or p55 (IL-2Ra) and the /3 chain or p70 (IL-2RB). Molecular cloning and expression of these two subunits demonstrate that IL-2Ra corresponds to the low-affinity receptor (2-4), whereas IL-2RP is

Small-Molecule Inhibitors of IL-2/IL-2R: Lessons Learned and Applied

“…IL-2 is a compact globular protein, composed of four tightly packed a-helices adopting a down-down-up-up configuration (cytokine fold) common to many interleukins and (Bazan 1990;Rozwarski et al 1994). A single disulfide bond establishes a covalent link between helix A and the middle of a 13 residue stretch of extended peptide preceding helix D. Site-specific mutagenesis identified a set of surface residues (Lys 35, Arg 38, Phe 42, Lys 43) critical for receptor binding; these residues lie on a concave face of IL-2 whose character (hydrophobic and basic) and location were consistent with a ligand-receptor hotspot for the PPI (Sauve et al 1991).…”

Hematopoietic cytokines: Similarities and differences in the structures, with implications for receptor binding

“…Site-directed mutagenesis experiments on IL-2 suggested that Asp 20 could play similar role for that cytokine (Zurawski et al, 1990;Sauve et al, 1991), and Imler et al (1992) identified the region of the receptor involved directly in this interaction. That residue was aligned with Glu 21 in GM-CSF (Shanafelt et al, 1991).…”

Binding of small molecules to an adaptive protein–protein interface

“…2a). Previous mutational work (17)(18)(19) has identified residues important for binding of IL-2 to the IL-2 receptor (a ''hot spot''). Even though Compound 1 was not designed to bind IL-2, it does bind directly over the region of IL-2 that is functionally critical for binding to IL-2R␣ (Fig.…”

“…None of the cysteine substitutions caused a substantial change in the ability of IL-2 to bind to IL-2R␤ (Table 2), suggesting they did not significantly perturb the conformation of the protein. Some of the mutants (Y45C, L72C, and K43C) did interfere with IL-2R␣ binding, as anticipated from previous mutagenesis studies that define the IL-2 hot spot (17)(18)(19). Each cysteine variant was screened against a 7,000-compound tethering library (6) in pools of 10 compounds each.…”