Interaction of the isolated transmembrane domain of diphtheria toxin with membranes.
Insertion of diphtheria toxin's T (transmembrane) domain into the endosomal membrane under acidic conditions is known to promote translocation of its catalytic domain across the membrane and into the cytosol. The T domain, a cysteine-free bundle of alpha-helices, was expressed as a discrete protein in Escherichia coli and purified. The isolated domain was stable and largely monomeric at pH 8.0. Like the holotoxin it bound the hydrophobic fluorophore, 2-p-toluidinylnaphthalene 6-sulfonate, upon acidification, but the transition pH was higher than with the holotoxin (pH 5.6 vs 5.1) and broader, reflecting the absence of interdomain interactions. The domain also permeabilized large unilamellar vesicles under acidic conditions, as demonstrated by release of entrapped solutes. Mutant forms of T domain, each with a single residue replaced by cysteine, were derivatized with a thiol-reactive nitroxide-containing spin label and analyzed by electron paramagnetic resonance (EPR). EPR spectra and solvent accessibilities of the labels at pH 8.0 were consistent with the environments predicted from the toxin's crystallographic structure. Acidification in the presence of large unilamellar vesicles caused a nitroxide label at position 332 on helix TH8 to move from a buried site in the water soluble state to a lipid-exposed surface site at a depth of approximately 15 A within the bilayer. This is consistent with the concept that the TH8-TH9 helix pair inserts into the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)
Zhan
H
H
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Oh
K J
KJ
Shin
Y K
YK
Hubbell
W L
WL
Collier
R J
RJ
D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D004167:Diphtheria Toxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011487:Protein Conformation
D017931:DNA Primers; D004167:Diphtheria Toxin