Structure/function analysis of the transmembrane domain of DAB₃₈₉- interleukin-2, an interleukin-2 receptor-targeted fusion toxin. The amphipathic helical region of the transmembrane domain is essential for the efficient delivery of the catalytic domain to the cytosol of target cells

Cassette and deletion mutagenesis were used to analyze the function of the amphipathic α-helices in the transmembrane domain of DAB₃₈₉-interleukin- 2 (IL-2), a fusion protein which is targeted to the interleukin-2 receptor. We demonstrate that the in-frame deletion of 60 amino acids, from Asn²⁰⁴ to Glu²⁶³ in DAB₃₈₉-IL-2, results in complete loss of cytotoxic activity, whereas when the amphipathic regions from Asp²⁰⁸ to Ser²²⁰ and Ala²⁴⁴ to His²⁵⁸ are replaced with idealized amphipathic helices composed of repeating Glu, Lys, and Leu residues, the mutant fusion toxin has low but detectable activity. DAB₃₈₉-IL-2 and both variants form channels in artificial phospholipid bilayers with conductances identical to those formed by diphtheria toxin. Both mutant fusion toxins bind to the high affinity IL-2 receptor with affinities similar to that of DAB₃₈₉-IL-2. The fact that these mutants have markedly reduced or absent cytotoxic activity, but possess 'wild type' catalytic activity, binding affinities, and channel conductances, suggests the existence of a step in the intoxication pathway, defective in the mutants, which occurs after DAB₃₈₉-IL-2 binds to the IL- 2 receptor. It is unknown whether this step occurs prior or subsequent to channel formation, but it is essential for the efficient delivery of the ADP- ribosyltransferase from DAB₃₈₉-IL-2 to the cytosol of target cells.