title: Amino acid residues in the N-terminal region of the BinB subunit of Lysinibacillus sphaericus binary toxin play a critical role during receptor binding and membrane insertion.
abstract: The binary toxin produced by Lysinibacillus sphaericus is composed of BinA and BinB subunits that work together in governing toxicity against mosquito larvae. BinA is proposed to be important for toxicity, whereas BinB has been shown to act as a specific receptor-binding component. The precise function of both subunits, however, is not well established. Here, we investigated the function of the N-terminal region of BinB subunit initially by introducing triple alanine substitutions at positions 35PEI37 and 41FYN43. Both block mutations abolished the larvicidal activity. Single point mutations (P35A, E36A, I37A, F41A, Y42A, N43A) were generated in order to identify amino acids that are critical for the toxin activity. Mosquito-larvicidal activity was significantly reduced in P35A, E36A, F41A and Y42A mutants. However, these mutants retained ability to form in vitro interaction with the BinA counterpart. Immunohistochemistry analysis revealed that P35A, F41A and N43A bind to the larval midgut membrane at comparable levels to that of the wild type BinB. In contrast, greatly reduced binding activity was observed in the Y42A, suggesting an important role of this residue in receptor binding. Alanine substitution at P35 resulted in a marked decrease in membrane penetration, indicating its functional importance for the membrane insertion. These results suggest the important roles of the N-terminal region of BinB in both the receptor recognition and the membrane interaction.
authors.affiliation: Institute of Molecular Biosciences, Mahidol University, Salaya Campus, 25/25 Putthamonthon 4 Road, Nakhon Pathom 73170, Thailand.
authors.forename: Kamonnut
authors.initials: K
authors.lastname: Singkhamanan
authors.forename: Boonhiang
authors.initials: B
authors.lastname: Promdonkoy
authors.forename: Toemsak
authors.initials: T
authors.lastname: Srikhirin
authors.forename: Panadda
authors.initials: P
authors.lastname: Boonserm
mesh_terms: D000596:Amino Acids; D000818:Animals; D001406:Bacillaceae / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D009033:Culicidae; D005798:Genes, Bacterial; D007814:Larva; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics; D011956:Receptors, Cell Surface / Q000378:metabolism; D020539:Sequence Analysis, Protein
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D021122:Protein Subunits; D011956:Receptors, Cell Surface
keywords: BinB; Binary toxin; Lysinibacillus sphaericus; Membrane penetration; Mutagenesis; Receptor binding