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 alpha-helices in the transmembrane domain of DAB389-i nterleukin-2 (IL-2), a fusion protein which is targeted to the interle ukin-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 Asp2 08 to Ser220 and Ala244 to His258 are replaced with idealized amphipat hic helices composed of repeating Glu, Lys, and Leu residues, the muta nt fusion toxin has low but detectable activity. DAB389-IL-2 and both variants form channels in artificial phospholipid bilayers with conduc tances identical to those formed by diphtheria toxin. Both mutant fusi on toxins bind to the high affinity IL-2 receptor with affinities simi lar to that of DAB389-IL-2. The fact that these mutants have markedly reduced or absent cytotoxic activity, but possess ''wild type'' cataly tic activity, binding affinities, and channel conductances, suggests t he existence of a step in the intoxication pathway, defective in the m utants, which occurs after DAB389-IL-2 binds to the IL-2 receptor. It is unknown whether this step occurs prior or subsequent to channel for mation, but it is essential for the efficient delivery of the ADPL-rib osyltransferase from DAB389-IL-2 to the cytosol of target cells.