Interleukin-2 (IL-2) is a 133 amino acid alpha-helical protein secreted by activated T-cells. Combinatorial cassette mutagenesis was used to investigate the functional role of a continuous five amino acid region of IL-2 suspected to interact with the intermediate-affinity IL-2 receptor. A limited random library of IL-2 mutants was constructed in which residues 17-21 (Leu-Leu-Leu-Asp-Leu) were simultaneously mutated. The proteins were produced in an Escherichia coli expression system and screened in a biological assay for their ability to mediate the proliferation of a murine IL-2-dependent cell line. From the over 2600 clones examined, only 42 exhibited significant activity, confirming the functional importance of this region. Selected clones were purified and further characterized by biological and receptor binding assays. Viewed in the context of the recently revised 2.5-A crystal structure for IL-2, these results suggest the following conclusions: both Asp20 and Leu21, as shown by their sensitivity to mutation, are the functionally more important residues in this region, but for different reasons. Asp20 is solvent-accessible and likely plays a direct receptor contact role as previous studies have indicated. Leu21, in contrast, is completely buried in the hydrophobic core of the protein. Substitutions at this position, even a conservative Leu-->Val substitution, were found to perturb the precise hydrophobic packing arrangements that are critical for activity, resulting in a significant loss of function. In addition, one of the analogs identified in the screen was found to be 2-3 times more potent than the wild-type protein.

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