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Conformational switching of the diphtheria toxin T domain.

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.

SUBMITTER: Rodnin MV

PROVIDER: S-EPMC3042302 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Conformational switching of the diphtheria toxin T domain.

Rodnin Mykola V MV Kyrychenko Alexander A Kienker Paul P Sharma Onkar O Posokhov Yevgen O YO Collier R John RJ Finkelstein Alan A Ladokhin Alexey S AS

Journal of molecular biology 20100721 1

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 struc ...[more]

PMID: 20654627

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Project description:Diphtheria toxin translocation (T) domain undergoes conformational changes in acidic solution and associates with the lipid membranes, followed by refolding and transmembrane insertion of two nonpolar helices. This process is an essential step in delivery of the toxic catalytic domain of the diphtheria toxin to the infected cell, yet its molecular determinants are poorly characterized and understood. Therefore, an atomistic model of the T-domain-membrane interaction is needed to help characterize factors responsible for such association. In this work, we present atomistic model structures of T-domain membrane-bound conformations and investigate structural factors responsible for T-domain affinity with the lipid bilayer in acidic solution using all-atom molecular dynamics (MD) simulations. The initial models of the protein conformations and protein-membrane association that serve as starting points in the present work were developed using atomistic simulations of partial unfolding of the T-domain in acidic solution (Kurnikov, I. V.; et al. J. Mol. Biol. 2013, 425, 2752-2764), and coarse-grained simulations of the T-domain association with the membranes of various compositions (Flores-Canales, J. C.; et al. J. Membr. Biol. 2015, 248, 529-543). In this work we present atomistic level modeling of two distinct configurations of the T-domain in association with the anionic lipid bilayer. In microsecond-long MD simulations both conformations retain their compact structure and gradually penetrate deeper into the bilayer interface. One membrane-bound conformation is stabilized by the protein contacts with the lipid hydrophobic core. The second modeled conformation is initially inserted less deeply and forms multiple contacts with the lipid at the interface (headgroup) region. Such contacts are formed by the charged and hydrophilic groups of partially unfolded terminal helixes and loops. Neutralization of the acidic residues at the membrane interface allows for deeper insertion of the protein and reorientation of the protein at the membrane interface, which corroborates that acidic residue protonation as well as presence of the anionic lipids may play a role in the membrane association and further membrane insertion of the T-domain as implicated in experiments. All simulations reported in this work were performed using AMBER force-field on Anton supercomputer. To perform these reported simulations, we developed and carefully tested a force-field for the anionic 1-palmitoyl-2-oleoyl-phosphatidyl-glycerol (POPG) lipid, compatible with the Amber 99SB force-field and stable in microsecond-long MD simulations in isothermal-isobaric ensemble.

Dataset Information

Conformational switching of the diphtheria toxin T domain.

Publications

Conformational switching of the diphtheria toxin T domain.

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