Department of Anesthesiology

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Engineering a pH responsive pore forming protein.

TitleEngineering a pH responsive pore forming protein.
Publication TypeJournal Article
Year of Publication2017
AuthorsKisovec M, Rezelj S, Knap P, Cajnko MMojca, Caserman S, Flašker A, Žnidaršič N, Repič M, Mavri J, Ruan Y, Scheuring S, Podobnik M, Anderluh G
JournalSci Rep
Volume7
Pagination42231
Date Published2017 Feb 08
ISSN2045-2322
Abstract

Listeriolysin O (LLO) is a cytolysin capable of forming pores in cholesterol-rich lipid membranes of host cells. It is conveniently suited for engineering a pH-governed responsiveness, due to a pH sensor identified in its structure that was shown before to affect its stability. Here we introduced a new level of control of its hemolytic activity by making a variant with hemolytic activity that was pH-dependent. Based on detailed structural analysis coupled with molecular dynamics and mutational analysis, we found that the bulky side chain of Tyr406 allosterically affects the pH sensor. Molecular dynamics simulation further suggested which other amino acid residues may also allosterically influence the pH-sensor. LLO was engineered to the point where it can, in a pH-regulated manner, perforate artificial and cellular membranes. The single mutant Tyr406Ala bound to membranes and oligomerized similarly to the wild-type LLO, however, the final membrane insertion step was pH-affected by the introduced mutation. We show that the mutant toxin can be activated at the surface of artificial membranes or living cells by a single wash with slightly acidic pH buffer. Y406A mutant has a high potential in development of novel nanobiotechnological applications such as controlled release of substances or as a sensor of environmental pH.

DOI10.1038/srep42231
Alternate JournalSci Rep
PubMed ID28176876
PubMed Central IDPMC5296754
Grant List310080 / / European Research Council / International