Engineering a pH responsive pore forming protein.
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.
Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.
Matic
M
Kisovec
Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.
Saša
S
Rezelj
Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.
Primož
P
Knap
Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.
Miša Mojca
MM
Cajnko
Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.
Simon
S
Caserman
Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.
Ajda
A
Flašker
Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia.
Nada
N
Žnidaršič
Department of Computational Biochemistry and Drug Design, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.
Matej
M
Repič
Department of Computational Biochemistry and Drug Design, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.
Janez
J
Mavri
U1006 Institut National de la Santé et de la Recherche Médicale (INSERM), Université Aix-Marseille, Parc Scientifique et Technologique de Luminy, 163 avenue de Luminy, 13009, Marseille, France.
Yi
Y
Ruan
U1006 Institut National de la Santé et de la Recherche Médicale (INSERM), Université Aix-Marseille, Parc Scientifique et Technologique de Luminy, 163 avenue de Luminy, 13009, Marseille, France.
Simon
S
Scheuring
Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.
Marjetka
M
Podobnik
Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.
Gregor
G
Anderluh
D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D018938:Caco-2 Cells; D002463:Cell Membrane Permeability; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008563:Membrane Lipids / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D052899:Pore Forming Cytotoxic Proteins / Q000737:chemistry / Q000378:metabolism*; D000072417:Protein Domains; D015202:Protein Engineering / Q000379:methods*; D012756:Sheep
D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D008563:Membrane Lipids; D050505:Mutant Proteins; D052899:Pore Forming Cytotoxic Proteins; C038705:hlyA protein, Listeria monocytogenes