Role of the alpha Clamp in the Protein Translocation Mechanism of Anthrax Toxin

摘要

Membrane-embedded molecular machines are utilized to move water-soluble proteins across these barriers. Anthrax toxin forms one such machine through the self-assembly of its three component proteins protective antigen (PA), lethal factor, and edema factor. Upon endocytosis into host cells, acidification of the endosome induces PA to form a membrane-inserted channel, which unfolds lethal factor and edema factor and translocates them into the host cytosol. Translocation is driven by the proton motive force, composed of the chemical potential, the proton gradient (Delta pH), and the membrane potential (Delta psi). A crystal structure of the lethal toxin core complex revealed an "alpha clamp" structure that binds to substrate helices nonspecifically. Here, we test the hypothesis that, through the recognition of unfolding helical structure, the alpha clamp can accelerate the rate of translocation. We produced a synthetic PA mutant in which an alpha helix was crosslinked into the alpha clamp to block its function. This synthetic construct impairs translocation by raising a yet uncharacterized translocation barrier shown to be much less force dependent than the known unfolding barrier. We also report that the alpha clamp more stably binds substrates that can form helices than those, such as polyproline, that cannot. Hence, the alpha clamp recognizes substrates by a general shape-complementarity mechanism. Substrates that are incapable of forming compact secondary structure (due to the introduction of a polyproline track) are severely deficient for translocation. Therefore, the a clamp and its recognition of helical structure in the translocating substrate play key roles in the molecular mechanism of protein translocation. (c) 2015 The Authors. Published by Elsevier Ltd.