Narahari Akkaladevi, Srayanta Mukherjee, Hiroo Katayama, Blythe Janowiak, Deepa Patel, Edward P. Gogol, Bradley L. Pentelute, R. John Collier, Mark T. Fisher*
J. Membr. Biol., 2015 (with Fisher lab KUMC)
13 January 2015
Bacterial toxin or viral entry into the cell often requires cell surface binding and endocytosis. The endo- somal acidification induces a limited unfolding/refolding and membrane insertion reaction of the soluble toxins or viral proteins into their translocation competent or mem- brane 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 immo- bilized 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.