MUTAGENIC ANALYSIS OF A RECEPTOR CONTACT SITE ON INTERLEUKIN-2 - PREPARATION OF AN IL-2 ANALOG WITH INCREASED POTENCY

Interleukin-2 (IL-2) is a 133 amino acid alpha-helical protein secrete d by activated T-cells. Combinatorial cassette; mutagenesis was used t o investigate the functional role of a contiguous five amino acid regi on of IL-2 suspected to interact with the intermediate-affinity IL-2 r eceptor. A limited random library of IL-2 mutants was constructed in w hich residues 17-21 (Leu-Leu-Leu-Asp-Leu) were simultaneously mutated. The proteins were produced in an Escherichia coli expression system a nd screened in a biological assay for their ability to mediate the pro liferation of a murine IL-1-dependent cell line. From the over 2600 cl ones examined, only 42 exhibited significant activity, confirming the functional importance of this region. Selected clones were purified an d further characterized by biological and receptor binding assays. Vie wed in the context of the recently revised 2.5-Angstrom crystal struct ure for IL-2, these results suggest the following conclusions: both As p20 and Leu21, as shown by their sensitivity to mutation, are the func tionally more important residues in this region, but for different rea sons. Asp20 is solvent-accessible and likely plays a direct receptor c ontact role as previous studies have indicated. Leu21, in contrast, is completely buried in the hydrophobic core of the protein. Substitutio ns at this position, even a conservative Leu --> Val substitution, wer e found to perturb the precise hydrophobic packing arrangements that a re critical for activity, resulting In addition, one of the analogs id entified in the screen was found to be 2-3 times more potent than the wild-type protein.