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Membrane translocation assay based on proteolytic cleavage: application to diphtheria toxin T domain.

ABSTRACT: The function of diphtheria toxin translocation (T) domain is to transfer the catalytic domain across the endosomal membrane upon acidification. The goal of this study was to develop and apply an in vitro functional assay for T domain activity, suitable for investigation of structure-function relationships of translocation across lipid bilayers of various compositions. Traditionally, T domain activity in vitro is estimated by measuring either conductance in planar lipid bilayers or the release of fluorescent markers from lipid vesicles. While an in vivo cell death assay is the most relevant to physiological function, it cannot be applied to studying the effects of pH or membrane lipid composition on translocation. Here we suggest an assay based on cleavage of the N-terminal part of T domain upon translocation into protease-loaded vesicles. A series of control experiment was used to confirm that cleavage occurs inside the vesicle and not as the result of vesicle disruption. Translocation of the N-terminus of the T domain is shown to require the presence of a critical fraction of anionic lipids, which is consistent with our previous biophysical measurements of insertion. Application of the proposed assay to a series of T domain mutants correlated well with the results of cytotoxicity assay.

SUBMITTER: Rodnin MV

PROVIDER: S-EPMC4259893 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Membrane translocation assay based on proteolytic cleavage: application to diphtheria toxin T domain.

Rodnin Mykola V MV Ladokhin Alexey S AS

Biochimica et biophysica acta 20141005 1 Pt A

The function of diphtheria toxin translocation (T) domain is to transfer the catalytic domain across the endosomal membrane upon acidification. The goal of this study was to develop and apply an in vitro functional assay for T domain activity, suitable for investigation of structure-function relationships of translocation across lipid bilayers of various compositions. Traditionally, T domain activity in vitro is estimated by measuring either conductance in planar lipid bilayers or the release of ...[more]

PMID: 25291602

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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

Membrane translocation assay based on proteolytic cleavage: application to diphtheria toxin T domain.

Publications

Membrane translocation assay based on proteolytic cleavage: application to diphtheria toxin T domain.

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