Alanine-scanning mutagenesis of α-helix D segment of interleukin-13 reveals new functionally important residues of the cytokine

We documented that α-helices A, C, and D in human interleukin-13 (IL13) participate in interaction with its respective receptors. We hypothesized that α-helix D is the site II of the cytokine that binds IL13Rα1, a component of the normal tissue heterodimeric signaling IL13/4 receptor (IL13/4R), and that α-helix D independently binds a monomeric IL13Rα2 receptor, which is a nonsignaling glioma-restricted receptor for IL13. Therefore, we alanine-scanned mutagenized helix D of IL13 to identify the residues involved in the respective receptors interaction. Recombinant muteins of IL13 were produced in Escherichia coli, and their structural integrity and identity were verified. The alanine mutants were tested in functional cellular assays, in which IL13 interaction with IL13Rα2 (glioma cells) or an ability to functionally stimulate IL13/4R (TF-1 cells) were examined, and also in binding assays. We found that residues 105, 106, and 109 of the D-helix of IL13 are responsible for interacting with the glioma-associated receptor. Moreover, glutamic acids at positions 92 and 110, and leucine at position 104 was found to be important for IL13/4R stimulation. Thus, α-helix D of IL13 is the primary site responsible for interaction with the IL13 binding proteins. We propose a model that illustrates the binding mode of IL13 with cancer-related IL13Rα2 and physiological IL13/4R.