Analyzing topography of membrane-inserted diphtheria toxin T domain using BODIPY-streptavidin: at low pH, helices 8 and 9 form a transmembrane hairpin but helices 5-7 form stable nonclassical inserted segments on the cis side of the bilayer.
Low pH-induced membrane insertion by diphtheria toxin T domain is crucial for A chain translocation into the cytoplasm. To define the membrane topography of the T domain, the exposure of biotinylated Cys residues to the cis and trans bilayer surfaces was examined using model membrane vesicles containing a deeply inserted T domain. To do this, the reactivity of biotin with external and vesicle-entrapped BODIPY-labeled streptavidin was measured. The T domain was found to insert with roughly 70-80% of the molecules in the physiologically relevant orientation. In this orientation, residue 349, located in the loop between hydrophobic helices 8 and 9, was exposed to the trans side of the bilayer, while other solution-exposed residues along the hydrophobic helices 5-9 region of the T domain located near the cis surface. A protocol developed to detect the movement of residues back and forth across the membranes demonstrated that T domain sequences did not rapidly equilibrate between the cis and the trans sides of the bilayer. Binding streptavidin to biotinylated residues prior to membrane insertion only inhibited T domain pore formation for residues in the loop between helices 8 and 9. Pore formation experiments used an approach avoiding interference from transient membrane defects/leakage that may occur upon the initial insertion of protein. Combined, these results indicate that at low pH hydrophobic helices 8 and 9 form a transmembrane hairpin, while hydrophobic helices 5-7 form a nonclassical deeply inserted nontransmembraneous state. We propose that this represents a novel pre-translocation state that is distinct from a previously defined post-translocation state.
Rosconi
Michael P
MP
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, New York 11794-5215, USA.
Zhao
Gang
G
London
Erwin
E
D000595:Amino Acid Sequence; D019921:Biotinylation; D001896:Boron Compounds; D002462:Cell Membrane / Q000378:metabolism*; D002463:Cell Membrane Permeability; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000378:metabolism; D005470:Fluorometry; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000378:metabolism; D008958:Models, Molecular; D010449:Peptide Mapping / Q000379:methods*; D017433:Protein Structure, Secondary; D021381:Protein Transport; D019809:Streptavidin
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D004167:Diphtheria Toxin; D008051:Lipid Bilayers; D019809:Streptavidin