TY - JOUR
T1 - Conformational Switching of the Diphtheria Toxin T Domain
AU - Rodnin, Mykola V.
AU - Kyrychenko, Alexander
AU - Kienker, Paul
AU - Sharma, Onkar
AU - Posokhov, Yevgen O.
AU - Collier, R. John
AU - Finkelstein, Alan
AU - Ladokhin, Alexey S.
N1 - Funding Information:
We are grateful to Dr. S. H. White for his valuable comments and to Mr. M. A. Myers for his editorial assistance. This research was supported by NIH grants GM-069783 (A.S.L), GM-29210 (A.F.) and AI-022021 (R.J.C).
PY - 2010/9
Y1 - 2010/9
N2 - 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.
AB - 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.
KW - Conformational switch
KW - Histidine protonation
KW - Lipid bilayer insertion
KW - Membrane protein folding
KW - Tryptophan fluorescence
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U2 - 10.1016/j.jmb.2010.07.024
DO - 10.1016/j.jmb.2010.07.024
M3 - Article
C2 - 20654627
AN - SCOPUS:77956172558
SN - 0022-2836
VL - 402
SP - 1
EP - 7
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 1
ER -