Importance of polarity of the α4-α5 loop residue-Asn166 in the pore-forming domain of the Bacillus thuringiensis Cry4Ba toxin: Implications for ion permeation and pore opening

Bacillus thuringiensis Cry4Ba toxin is lethal to mosquito-larvae by forming ion-permeable pores in the target midgut cell membrane. Previously, the polarity of Asn(166) located within the alpha 4-alpha 5 loop composing the Cry4Ba pore-forming domain was shown to be crucial for larvicidal activity. Here, structurally stable-mutant toxins of both larvicidal-active (N166D) and inactive (N166A and N1661) mutants were FPLC-purified and characterized for their relative activities in liposornal similar to membrane permeation and single-channel formation. Similar to the 65-kDa trypsin-activated wild-type toxin, the N166D bio-active mutant toxin was still capable of releasing entrapped calcein from lipid vesicles. Conversely, the two other bio-inactive mutants showed a dramatic decrease in causing membrane permeation. When the N166D mutant was incorporated into planar lipid bilayers (under symmetrical conditions at 150 mM KCl, pH 8.5), it produced single-channel currents with a maximum conductance of about 425 pS comparable to the wild-type toxin. However, maximum conductances for single K+-channels formed by both bio-inactive mutants (N1661 and N166A) were reduced to approximately 165-205 pS. Structural dynamics of 60-ns simulations of a trimeric alpha 4-alpha 5 pore model in a fully hydrated-DMPC system revealed that an open-pore structure could be observed only for the simulated pores of the wild type and N166D. Additionally, the number of lipid molecules interacting with both wild-type and N166D pores is relatively higher than those of N166A and N1661 pores. Altogether, our results further signify that the polarity at the alpha 4-alpha 5 loop residue similar to Asn(166) is directly involved in ion permeation through the Cry4Ba toxin-induced ionic pore and pore opening at the membrane-water interface. (C) 2013 Elsevier B.V. All rights reserved.