Conformational switching of the diphtheria toxin T domain.

Journal of Molecular Biology
Mykola V RodninAlexey S Ladokhin

Abstract

The diphtheria toxin T domain translocates the catalytic C domain across the endosomal membrane in response to acidification. To elucidate the role of histidine protonation in modulating pH-dependent membrane action of the T domain, we have used site-directed mutagenesis coupled with spectroscopic and physiological assays. Replacement of H257 with an arginine (but not with a glutamine) resulted in dramatic unfolding of the protein at neutral pH, accompanied by a substantial loss of helical structure and greatly increased exposure of the buried residues W206 and W281. This unfolding and spectral shift could be reversed by the interaction of the H257R mutant with model lipid membranes. Remarkably, this greatly unfolded mutant exhibited wild-type-like activity in channel formation, N-terminus translocation, and cytotoxicity assays. Moreover, membrane permeabilization caused by the H257R mutant occurs already at pH 6, where wild type protein is inactive. We conclude that protonation of H257 acts as a major component of the pH-dependent conformational switch, resulting in destabilization of the folded structure in solution and thereby promoting the initial membrane interactions necessary for translocation.

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Citations

Oct 13, 2012·Chemical Reviews·Ekaterina M Nestorovich, Sergey M Bezrukov
Mar 5, 2013·Annual Review of Biophysics·André SchönichenDiane L Barber
Apr 30, 2013·Biochemistry·Mauricio Vargas-UribeAlexey S Ladokhin
Oct 8, 2014·Biochimica Et Biophysica Acta·Mykola V Rodnin, Alexey S Ladokhin
Mar 19, 2014·The FEBS Journal·Oneda LekaGiuseppe Zanotti
Feb 5, 2015·The Journal of Membrane Biology·Jose C Flores-CanalesMaria Kurnikova
Oct 5, 2014·The Journal of Membrane Biology·Mauricio Vargas-UribeAlexey S Ladokhin
May 8, 2013·Journal of Molecular Biology·Igor V KurnikovAlexey S Ladokhin
Sep 18, 2012·Cell Host & Microbe·Mirko BischofbergerF Gisou van der Goot
Nov 3, 2016·Biophysical Journal·Mykola V RodninAlexey S Ladokhin
May 27, 2017·Critical Reviews in Biochemistry and Molecular Biology·Kathleen E OrrellRoman A Melnyk
Apr 4, 2013·Bioorganicheskaia khimiia·S I RomaniukS V Komisarenko
Oct 1, 2015·Methods and Applications in Fluorescence·Alexander Kyrychenko
Jul 22, 2016·The Journal of Biological Chemistry·Ashley N MartfeldRoger E Koeppe
Feb 23, 2021·Journal of Drug Targeting·Mohammad Reza KhirehgeshBahman Akbari
Apr 21, 2019·Biochimica Et Biophysica Acta. Proteins and Proteomics·Victor Vasquez-MontesAlexey S Ladokhin

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