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.

Lysenin Channels as Sensors for Ions and Molecules

“…The ability of pore-forming proteins and peptides to establish conducting pathways between two sides of a lipid membrane was exploited for decades for numerous analytical applications [1][2][3][4][5][6][7][8][9]. The most common sensing principle relies on measuring changes in the ionic currents elicited by specific and non-specific interactions between analytes of interest and wild-type or engineered protein channels [10][11][12][13][14][15][16][17].…”

NMR Study of Solvation Effect on the Geometry of Proton-Bound Homodimers of Increasing Size

“…[9][10][11] At the current stage of knowledge, it is possible to predict the effect of a mutation on the fate of a particular biosystem, to prove this prediction experimentally, and to explain it theoretically. 12 However, for many formally simple systems experimental results have not been reproduced in silico at a reasonable approximation; the strength of dispersive interactions in solution, 13 spectroscopic signatures of protonated water clusters 14,15 and hydrated hydroxyl anion, 16 the structure of water-base complexes in aprotic solutions 17 and amorphous solids [18][19][20] are among such problems. In such cases, the measured spectral parameters have to be assigned to a certain geometry or energy using empirical correlations.…”

Cytotoxic Activity of LLO Y406A Is Targeted to the Plasma Membrane of Cancer Urothelial Cells

“…To date, wt LLO activity has been studied in a variety of other cell types, including colon epithelial cells Caco-2 and HT-29/B6, embryonic kidney cells HEK 293, human brain endothelial cells, and Jurkat cells, each without comparing the response of wt LLO in cancer vs. normal cells [ 34 , 35 , 36 , 37 , 38 ]. Herein, we initially planned to use LLO Y406A, designed for activity at a narrow pH optimum of 5–6 [ 11 ] to prevent pore-forming activity at the plasma membrane with usually neutral pH surrounding, which should induce selective pore-forming activity in acidic endosomes. In RT4 cells, 0.5 µM LLO Y406A treatment decreased viability to 43%, whereas normal urothelial cells survived the same treatment with 95%.…”

“…LLO belongs to the family of cholesterol-dependent cytolysins that form pores in cholesterol-rich membranes [ 7 , 8 , 9 ]. Cholesterol concentration above 35% mol, which is typical for membrane rafts, is a prerequisite for efficient binding of LLO to the membranes [ 10 , 11 , 12 ]. A distinctive property of LLO is its pH-dependent stability, which exhibits an optimum at ~5.5, a condition found in late endosomes [ 13 , 14 ].…”

“…Many LLO mutants with different pH-dependent pore-forming, membrane-binding, and cholesterol-binding activities have been constructed [ 16 , 19 , 20 , 21 , 22 ]. The LLO mutant with a substitution (Try→Ala) at the site 406 (LLO Y406A) used in this study was shown to be capable to form transmembrane pores at pH 5.7, however, with only minimal pore-forming activity at the neutral pH in vitro [ 11 ]. Thus, LLO Y406A can be considered as a favorable membrane-damaging agent for selective action in membranes with an acidic environment.…”