pH-Triggered Conformational Switching of the Diphtheria Toxin T-Domain: The Roles of N-Terminal Histidines

• Published in: Journal of molecular biology 2013-08, Vol.425 (15), p.2752-2764
• Main Authors: Kurnikov, Igor V., Kyrychenko, Alexander, Flores-Canales, Jose C., Rodnin, Mykola V., Simakov, Nikolay, Vargas-Uribe, Mauricio, Posokhov, Yevgen O., Kurnikova, Maria, Ladokhin, Alexey S.
• Format: Article
• Language: English
• Subjects: acid-induced conformational change; acidification; Allosteric Regulation; Diphtheria Toxin - chemistry; Diphtheria Toxin - metabolism; fluorescence; fluorescence emission spectroscopy; histidine; histidine protonation; Hydrogen-Ion Concentration; hydrophobicity; membrane protein insertion; Models, Molecular; molecular dynamics; Molecular Dynamics Simulation; mutagenesis; Protein Conformation; Protein Folding - drug effects; Protein Transport - drug effects; Spectrometry, Fluorescence; Static Electricity; Thermodynamics
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2013.04.030

pH-induced conformational switching is essential for functioning of diphtheria toxin, which undergoes a membrane insertion/translocation transition triggered by endosomal acidification as a key step of cellular entry. In order to establish the sequence of molecular rearrangements and side-chain protonation accompanying the formation of the membrane-competent state of the toxin's translocation (T) domain, we have developed and applied an integrated approach that combines multiple techniques of computational chemistry [e.g., long-microsecond-range, all-atom molecular dynamics (MD) simulations; continuum electrostatics calculations; and thermodynamic integration (TI)] with several experimental techniques of fluorescence spectroscopy. TI calculations indicate that protonation of H257 causes the greatest destabilization of the native structure (6.9kcal/mol), which is consistent with our early mutagenesis results. Extensive equilibrium MD simulations with a combined length of over 8μs demonstrate that histidine protonation, while not accompanied by the loss of structural compactness of the T-domain, nevertheless results in substantial molecular rearrangements characterized by the partial loss of secondary structure due to unfolding of helices TH1 and TH2 and the loss of close contact between the C- and N-terminal segments. The structural changes accompanying the formation of the membrane-competent state ensure an easier exposure of the internal hydrophobic hairpin formed by helices TH8 and TH9, in preparation for its subsequent transmembrane insertion. [Display omitted] •Conformational switching is investigated by computational and experimental methods.•Microsecond MD simulations reveal conformational change upon histidine protonation.•TI is used to calculate free energy of histidine protonation.•Protonation of H257 ensures conformational changes.•Protonation of H223 protects from premature protonation of H257.