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Following Natures Lead: On the Construction of Membrane-Inserted Toxins in Lipid Bilayer Nanodiscs

Abstract

Bacterial toxin or viral entry into the cell often requires cell surface binding and endocytosis. The endosomal acidification induces a limited unfolding/refolding and membrane insertion reaction of the soluble toxins or viral proteins into their translocation competent or membrane inserted states. At the molecular level, the specific orientation and immobilization of the pre-transitioned toxin on the cell surface is often an important prerequisite prior to cell entry. We propose that structures of some toxin membrane insertion complexes may be observed through procedures where one rationally immobilizes the soluble toxin so that potential unfolding ↔ refolding transitions that occur prior to membrane insertion orientate away from the immobilization surface in the presence of lipid micelle pre-nanodisc structures. As a specific example, the immobilized prepore form of the anthrax toxin pore translocon or protective antigen can be transitioned, inserted into a model lipid membrane (nanodiscs), and released from the immobilized support in its membrane solubilized form. This particular strategy, although unconventional, is a useful procedure for generating pure membrane-inserted toxins in nanodiscs for electron microscopy structural analysis. In addition, generating a similar immobilized platform on label-free biosensor surfaces allows one to observe the kinetics of these acid-induced membrane insertion transitions. These platforms can facilitate the rational design of inhibitors that specifically target the toxin membrane insertion transitions that occur during endosomal acidification. This approach may lead to a new class of direct anti-toxin inhibitors.

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Acknowledgments

This work was supported grant funds from National Institutes of Health (NIH) R56 R56AI090085 (MTF), NIH R01AI090085 (MTF), and NIH SR37AI022021 (RJC). Some initial cryo-EM results presented in Figs. 3 and 4 were collected at the National Center for Imaging Macromolecules, supported by NIH Grant NIGMS P41 GM103832 (Wah Chiu). Additionally, some of the recombinant proteins used in the study were prepared in the Biomolecule Production Core of the New England Regional Center of Excellence (NERCE), supported by NIH Grant Number AI057159.

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Correspondence to Mark T. Fisher.

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Akkaladevi, N., Mukherjee, S., Katayama, H. et al. Following Natures Lead: On the Construction of Membrane-Inserted Toxins in Lipid Bilayer Nanodiscs. J Membrane Biol 248, 595–607 (2015). https://doi.org/10.1007/s00232-014-9768-3

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Keywords

  • Anthrax protective antigen
  • GroEL
  • Lipid nanodiscs
  • Biolayer interferometry
  • Electron microscopy