INTERACTION OF THE ISOLATED TRANSMEMBRANE DOMAIN OF DIPHTHERIA-TOXIN WITH MEMBRANES

Insertion of diphtheria toxin's T (transmembrane) domain into the endo somal membrane under acidic conditions is known to promote translocati on of its catalytic domain across the membrane and into the cytosol. T he T domain, a cysteine-free bundle of ol-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 bou nd the hydrophobic fluorophore, 2-p-toluidinylnaphthalene 6-sulfonate, upon acidification, but the transition pH was higher than with the ho lotoxin (pH 5.6 vs 5.1) and broader, reflecting the absence of interdo main interactions. The domain also permeabilized large unilamellar ves icles under acidic conditions, as demonstrated by release of entrapped solutes. Mutant forms of 7 domain, each with a single residue replace d by cysteine, were derivatized with a thiol-reactive nitroxide-contai ning 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 crystallog raphic structure. Acidification in the presence of large unilamellar v esicles 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 surfa ce site at a depth of similar to 15 Angstrom within the bilayer. This is consistent with the concept that the TH8-TH9 helix pair inserts int o the bilayer. Changes in the nitroxide at residue 351 suggest that th is side chain, located in the loop linking helices TH8 and TH9, moves to a buried site within the tertiary fold of a reorganized monomer or within an oligomeric structure formed in the membrane. These findings validate the isolated T domain as an object of investigation and demon strate the potential of site-directed spin labeling for elucidating th e structure of diphtheria toxin within membranes.