title: Hydrogen-deuterium exchange and mass spectrometry reveal the pH-dependent conformational changes of diphtheria toxin T domain.
abstract: The translocation (T) domain of diphtheria toxin plays a critical role in moving the catalytic domain across the endosomal membrane. Translocation/insertion is triggered by a decrease in pH in the endosome where conformational changes of T domain occur through several kinetic intermediates to yield a final trans-membrane form. High-resolution structural studies are only applicable to the static T-domain structure at physiological pH, and studies of the T-domain translocation pathway are hindered by the simultaneous presence of multiple conformations. Here, we report the application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) for the study of the pH-dependent conformational changes of the T domain in solution. Effects of pH on intrinsic HDX rates were deconvolved by converting the on-exchange times at low pH into times under our "standard condition" (pH 7.5). pH-Dependent HDX kinetic analysis of T domain clearly reveals the conformational transition from the native state (W-state) to a membrane-competent state (W(+)-state). The initial transition occurs at pH 6 and includes the destabilization of N-terminal helices accompanied by the separation between N- and C-terminal segments. The structural rearrangements accompanying the formation of the membrane-competent state expose a hydrophobic hairpin (TH8-9) to solvent, prepare it to insert into the membrane. At pH 5.5, the transition is complete, and the protein further unfolds, resulting in the exposure of its C-terminal hydrophobic TH8-9, leading to subsequent aggregation in the absence of membranes. This solution-based study complements high resolution crystal structures and provides a detailed understanding of the pH-dependent structural rearrangement and acid-induced oligomerization of T domain.
authors.affiliation: Department of Chemistry, Washington University , St. Louis, Missouri 63130, United States.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Mykola V
authors.initials: MV
authors.lastname: Rodnin
authors.forename: Alexey S
authors.initials: AS
authors.lastname: Ladokhin
authors.forename: Michael L
authors.initials: ML
authors.lastname: Gross
mesh_terms: D018360:Crystallography, X-Ray; D041961:Deuterium Exchange Measurement; D004167:Diphtheria Toxin / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D004167:Diphtheria Toxin