Structural and functional characterization of the proline-rich region within the [alpha]4-[alpha]5 loop of the Bacillus thuringiensis Cry4Aa toxin

• Main Author: Chompounoot Imtong
• Other Authors: Chanan Angsuthanasombat
• Language: English
• Published: Mahidol University. Mahidol University Library and Knowledge Center 2023
• Subjects: Bacillus thuringiensis; Endotoxins
• Online Access: https://repository.li.mahidol.ac.th/handle/123456789/89534
• Institution: Mahidol University

The long loop connecting transmembrane helices 4 and 5 of the Bacillus thuringiensis Cry4Aa toxin possesses unique structural features including a proline-rich sequence (Pro[superscript]193Pro[superscript]194_Pro[superscript]196) which was previously shown to be crucial for mosquito-larvicidal activity. In this study, the structural importance of the proline-rich region was further clarified. Initially, the three proline residues (Pro[superscript]193, Pro[superscript]194 and Pro[superscript]196) were substituted individually with valine, a hydrophobic residue with size similar to proline, and the resulting mutants were expressed in Escherichia coli cells at levels similar to that of the wild-type toxin. Bioassays demonstrated that E. coli cells expressing each mutant toxin exhibited a reduction in toxicity against Aedes aegypti mosquito larvae, suggesting an important role in toxin function of unique cyclic structure, not hydrophobic feature, of the loop proline residues. Molecular dynamics (MD) simulations of the 65-kDa Cry4Aa structure in 150 mM KCl solution revealed that the α4-α5 loop is substantially stable as it showed a low structural fluctuation with RMSF value of 1.2 A. When the α4-α5 loop flexibility was increased through P193G, P194G and P196G mutations, a decrease in larvicidal activity was also observed for all mutants, suggesting that loop rigidity provided by cyclic side chain of proline residues is functionally important. Further structural analysis via MD simulations revealed that only the most critical proline residue, Pro[superscript]193, at which mutations adversely affect toxin solubility is in close contact with several surrounding residues, thus playing an additional role in structural arrangement of the Cry4Aa toxin molecule. Altogether, present data suggest that structural stability of the Cry4Aa α4-α5 loop comprising the proline-rich sequence plays an essential role in the toxin function