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

Abstract

Bacillus thuringiensis Cry4Ba toxin is lethal to mosquito-larvae by forming ion-permeable pores in the target midgut cell membrane. Previously, the polarity of Asn166 located within the α4–α5 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 N166I) mutants were FPLC-purified and characterized for their relative activities in liposomal-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 150mM KCl, pH8.5), it produced single-channel currents with a maximum conductance of about 425pS comparable to the wild-type toxin. However, maximum conductances for single K+-channels formed by both bio-inactive mutants (N166I and N166A) were reduced to approximately 165–205pS. Structural dynamics of 60-ns simulations of a trimeric α4–α5 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 N166I pores. Altogether, our results further signify that the polarity at the α4–α5 loop residue—Asn166 is directly involved in ion permeation through the Cry4Ba toxin-induced ionic pore and pore opening at the membrane–water interface.