Engineering a pH responsive pore forming protein

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.
Document type :
Journal articles
Complete list of metadatas

Cited literature [42 references]  Display  Hide  Download

https://hal-amu.archives-ouvertes.fr/hal-01814064
Contributor : Administrateur Hal Amu <>
Submitted on : Tuesday, June 12, 2018 - 5:47:11 PM
Last modification on : Thursday, November 29, 2018 - 8:14:02 PM

File

srep42231.pdf
Publisher files allowed on an open archive

Licence


Distributed under a Creative Commons Attribution 4.0 International License

Identifiers

Collections

Citation

Matic Kisovec, Sasa Rezelj, Primoz Knap, Misa Mojca Cajnko, Simon Caserman, et al.. Engineering a pH responsive pore forming protein. Scientific Reports, Nature Publishing Group, 2017, 7, pp.42231. ⟨10.1038/srep42231⟩. ⟨hal-01814064⟩

Share

Metrics

Record views

163

Files downloads

60