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.
Highlights
Membranes[19] and oligomerizes into arc-like structures on the surface of the membranes[20,21]
LLO is especially interesting for engineering because of its naturally present pH dependent stability, which is important for its biological role
The unfolding leads to premature aggregation of the protein in solution in the absence of the membrane, and causes its inactivation[22,23]
Summary
LLO stability is dependent on pH and temperature[22,23,28]. A pH-sensor composed of three negatively charged amino acids (D208, E247, D320) was identified in D322 (Fig. 1). Adsorbed Y406A was about 10.5 nm in height, the extramembraneous part of the inserted protein is lower and protrudes only about 5.5 nm (Fig. 4d) These results altogether confirm that the mutant is able to interact with lipid membranes and that it can oligomerize into structures similar in shape to the LLO. We show here that the mutant is still well-soluble in aqueous solution, is slightly less stable than the wild-type protein due to an important structural role of the residue Y406, it maintains its membrane-binding capacity in a wide pH range It expresses a permeabilizing phenotype only at pH-values that are close to the acidic environment of the phagosomal vacuoles. LLO has proven to be amenable for engineering and its further developments will contribute to the growing need for protein nanopores used in sensing as well as in cell biology applications
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