His180 in the pore-lining α4 of the Bacillus thuringiensis Cry4Aa δ-endotoxin is crucial for structural arrangements of the α4-α5 transmembrane hairpin and hence biotoxicity

One proposed toxic mechanism of Bacillus thuringiensis Cry delta-endotoxins involves pore formation in target membranes by the alpha 4-alpha 5 transmembrane hairpin constituting their pore-forming domain. Here, nine selected charged and uncharged polar residues in the pore-lining alpha 4 of the Cry4Aa mosquito-active toxin were substituted with Ala. All mutant toxins, i.e., D169A, R171A, Q173A, H178A, Y179A, H180A, Q182A, N183A and E187A, were over-expressed in Escherichia coli as 130-kDa pmtoxin inclusions at levels comparable to the wild-type toxin. Bioassays against Aedes aegypti larvae revealed that only H178A and H180A mutants displayed a drastic reduction in biotoxicity, albeit almost complete insolubility observed for H178A, but not for H180A inclusions. Further mutagenic analysis showed that replacements of His(180) with charged (Arg, Lys, Asp, Glu), small uncharged polar (Ser, Cys) or small non-polar (Gly, Val) residues severely impaired the biotoxicity, unlike substitutions with relatively large uncharged (Asn, Gln, Leu) or aromatic (Phe, Tyr, Trp) residues. Similar to the trypsin-activated wild-type toxin, both bio-active and -inactive H180 mutants were still capable of releasing entrapped calcein from lipid vesicles and producing cation-selective channels with similar to 130-pS maximum conductance. Analysis of the Cry4Aa structure revealed the existence of a hydrophobic cavity near the critical His(180) side-chain. Analysis of simulated structures revealed that His(180)-to-smaller residue conversions create a gap disrupting such cavity's hydrophobicity and hence structural arrangements of the alpha 4-alpha 5 hairpin. Altogether, our data disclose a critical involvement in Cry4Aa-biotoxicity of His(180) exclusively present in the lumen-facing alpha 4 for providing proper environment for the alpha 4-alpha 5 hairpin prior to membrane-inserted pore formation.