Topography of diphtheria toxin A chain inserted into lipid vesicles.
The membrane-inserting T domain of diphtheria toxin aids the low-pH-triggered translocation of the catalytic A chain of the toxin across endosomal membranes. To evaluate the role of the isolated A chain in translocation, the topography of isolated A chain inserted into model membrane vesicles was investigated using a mixture either of dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG) or of dimyristoleoylphosphatidylcholine (DMoPC) and DOPG. The latter mixture was previously found to promote deep insertion of the T domain. A series of single Cys mutants along the A chain sequence were labeled with bimane or BODIPY groups. After A chain insertion into model membranes, the location of these groups within the lipid bilayer was determined via bimane fluorescence emission lambda(max), binding of externally added anti-BODIPY antibodies, and a novel technique involving the comparison of the quenching of bimane fluorescence by aqueous iodide and membrane-associated 10-doxylnonadecane. The results show that in both DOPC- and DMoPC-containing bilayers, membrane-inserted residues all along the A chain sequence occupy shallow locations that are relatively exposed to the external solution. There were only small differences between A chain topography in the two different types of lipid mixtures. However, the behavior of the A chain in the two different lipid mixtures was distinct in that it strongly oligomerized in DMoPC-containing vesicles as judged by Trp fluorescence. In addition, A chain selectively induced fusion of the DMoPC-containing vesicles, and this may aid oligomerization by increasing the A chain/vesicle ratio. Fusion may also explain why A chain also selectively induced leakage of the contents of DMoPC-containing vesicles. We propose that isolated A chain is unlikely to be inserted in a transmembrane orientation, and thus its interaction with the T domain is likely to be critical for properly orienting the A chain within the bilayer in a fashion that allows translocation.
Hayashibara
Masatoshi
M
Department of Biochemistry and Cell Biology, Stony Brook University, S.U.N.Y., Stony Brook, New York 11794-5215, USA.
London
Erwin
E
D009327:4-Chloro-7-nitrobenzofurazan / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry / Q000378:metabolism; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004227:Dithionite / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008561:Membrane Fusion; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010539:Permeability; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000235:genetics; D012996:Solutions; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D019086:Bridged Bicyclo Compounds, Heterocyclic; D004167:Diphtheria Toxin; D008051:Lipid Bilayers; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D021122:Protein Subunits; D012996:Solutions; C021149:bimanes; C029795:diphtheria toxin fragment A; D004227:Dithionite; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; D014364:Tryptophan; C017251:1,2-oleoylphosphatidylcholine; D009327:4-Chloro-7-nitrobenzofurazan; D003545:Cysteine; D004134:Dimyristoylphosphatidylcholine