Characterization of diphtheria toxin's catalytic domain interaction with lipid membranes.
In response to a low environmental pH and with the help of the B fragment (DTB) the catalytic domain of diphtheria toxin (DTA) crosses the endosomal membrane to inhibit protein synthesis. In this study, we investigated the interaction of DTA with lipid membranes by biochemical and biophysical approaches. Data obtained from proteinase K and trypsin digestion experiments of membrane-inserted DTA suggested that residues 134-157 may adopt a transmembrane orientation and residues 77-100 could be membrane-associated, adopting either a surface or a transmembrane orientation. Fourier transform infrared spectroscopy analysis (FTIR) was used to characterize the secondary and tertiary structure of DTA along its pathway, from the native secreted form at pH 7.2 to the refolded structure at neutral pH after interaction with and desorption from a lipid membrane. We found that the association of DTA with lipid membranes at low pH was characterized by an increase of beta-sheet structures and that the refolded structure at neutral pH after interaction with the membrane was identical to the native structure at the same pH. We also investigated the desorption of DTA from the membrane at neutral pH as a function of temperature. Although a complete desorption was observed at 37 degrees C, no desorption took place at 4 degrees C. A model of translocation involving the possibility that DTA might insert one or several transient transmembrane domains during translocation is discussed.
Structure et Fonction des Membranes Biologiques, CP 206/2, Université Libre de Bruxelles, Boulevard du Triomphe, B-1050 Brussels, Belgium.
Christian
C
Wolff
Ruddy
R
Wattiez
Jean-Marie
JM
Ruysschaert
Véronique
V
Cabiaux
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004167:Diphtheria Toxin / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008051:Lipid Bilayers; D017550:Spectroscopy, Fourier Transform Infrared
D004167:Diphtheria Toxin; D008051:Lipid Bilayers