title: Crucial role of H322 in folding of the diphtheria toxin T-domain into the open-channel state.
abstract: The translocation (T) domain plays a key role in the entry of diphtheria toxin into the cell. Upon endosomal acidification, the T-domain undergoes a series of conformational changes that lead to its membrane insertion and formation of a channel. Recently, we have reported that the triple replacement of C-terminal histidines H322, H323, and H372 with glutamines prevents the formation of open channels in planar lipid bilayers. Here, we report that this effect is primarily due to the mutation of H322. We further examine the relationship between the loss of functionality and membrane folding in a series of mutants with C-terminal histidine substitutions using spectroscopic assays. The membrane insertion pathway for the mutants differs from that of the wild type as revealed by the membrane-induced red shift of tryptophan fluorescence at pH 6.0-6.5. T-Domain mutants with replacements at H323 and H372, but not at H322, regain a wild-type-like spectroscopic signature upon further acidification. Circular dichroism measurements confirm that affected mutants misfold during insertion into vesicles. Conductance measurements reveal that substituting H322 dramatically reduces the numbers of properly folded channels in a planar bilayer, but the properties of the active channels appear to be unaltered. We propose that H322 plays an important role in the formation of open channels and is involved in guiding the proper insertion of the N-terminal region of the T-domain into the membrane.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center , Kansas City, Kansas 66160, United States.
authors.forename: Mauricio
authors.initials: M
authors.lastname: Vargas-Uribe
authors.forename: Mykola V
authors.initials: MV
authors.lastname: Rodnin
authors.forename: Paul
authors.initials: P
authors.lastname: Kienker
authors.forename: Alan
authors.initials: A
authors.lastname: Finkelstein
authors.forename: Alexey S
authors.initials: AS
authors.lastname: Ladokhin
mesh_terms: D001665:Binding Sites; D002942:Circular Dichroism; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D014364:Tryptophan / Q000235:genetics
chemical_list: D004167:Diphtheria Toxin; D008051:Lipid Bilayers; D006639:Histidine; D014364:Tryptophan