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

JOURNAL OF MOLECULAR BIOLOGY

卷:425

Article

Kurnikov, Igor V.¹  Kyrychenko, Alexander²  Flores-Canales, Jose C.¹  Rodnin, Mykola V.²  Simakov, Nikolay³  Vargas-Uribe, Mauricio²  Posokhov, Yevgen O.²  Kurnikova, Maria¹  Ladokhin, Alexey S.² 

[1] Carnegie Mellon Univ, Dept Chem, Pittsburgh, PA 15213 USA

[2] Univ Kansas, Med Ctr, Dept Biochem & Mol Biol, Kansas City, KS 66160 USA

[3] SUNY Buffalo, Ctr Computat Res, Buffalo, NY 14203 USA

关键词: acid-induced conformational change;    membrane protein insertion;    histidine protonation;    molecular dynamics;    fluorescence;   

DOI  :  10.1016/j.jmb.2013.04.030

来源: Elsevier

【 摘 要 】

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 (6.9 kcal/mol), which is consistent with our early mutagenesis results. Extensive equilibrium MD simulations with a combined length of over 8 mu s 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. (c) 2013 Elsevier Ltd. All rights reserved.

【 授权许可】

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