Engineering a pH responsive pore forming protein

• DOI: 10.1038/srep42231
• Publication Date: 2017-02-08T10:09:30Z
• Authors: Matic Kisovec; Saša Rezelj; Primož Knap; Miša Mojca Cajnko; Simon Caserman; Ajda Flašker; Nada Žnidaršič; Matej Repič; Janez Mavri; Yi Ruan; Simon Scheuring; Marjetka Podobnik; Gregor Anderluh
• Keywords: Cytolysin; Pore-forming toxin; Protein Engineering

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