The effects of helix breaking mutations in the diphtheria toxin transmembrane domain helix layers of the fusion toxin DAB389IL-2.
The fusion protein toxin DAB389IL-2 is composed of the catalytic and transmembrane domains of diphtheria toxin genetically linked to human interleukin 2 (IL-2). This fusion toxin is selectively toxic for eukaryotic cells which express the high-affinity form of the IL-2 receptor and the mechanism of intoxication parallels that of native diphtheria toxin. We used site-directed mutagenesis to introduce Pro residues into each of the three helical layers of the transmembrane domain. Although each of the mutations results in the complete loss of cytotoxic activity, individual mutants were found to vary with respect to channel formation in planar lipid bilayers, binding affinity and melting temperature. We propose that each of the three helix layers plays a critical role in the productive delivery of the catalytic domain to the cell cytosol.
Hu
H Y
HY
Evans Department of Clinical Research and Department of Medicine, Boston Medical Center, MA 02118, USA.
Huynh
P D
PD
Murphy
J R
JR
vanderSpek
J C
JC
D000595:Amino Acid Sequence; D002460:Cell Line; D004167:Diphtheria Toxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D000922:Immunotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007376:Interleukin-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D004167:Diphtheria Toxin; D000922:Immunotoxins; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C078456:denileukin diftitox