title: Role of tryptophan residues in toxicity of Cry1Ab toxin from Bacillus thuringiensis.
abstract: Bacillus thuringiensis produces insecticidal proteins (Cry protoxins) during the sporulation phase as parasporal crystals. During intoxication, the Cry protoxins must change from insoluble crystals into membrane-inserted toxins which form ionic pores. The structural changes of Cry toxins during oligomerization and insertion into the membrane are still unknown. The Cry1Ab toxin has nine tryptophan residues; seven are located in domain I, the pore-forming domain, and two are located in domain II, which is involved in receptor recognition. Eight Trp residues are highly conserved within the whole family of three-domain Cry proteins, suggesting an essential role for these residues in the structural folding and function of the toxin. In this work, we analyzed the role of Trp residues in the structure and function of Cry1Ab toxin. We replaced the Trp residues with phenylalanine or cysteine using site-directed mutagenesis. Our results show that W65 and W316 are important for insecticidal activity of the toxin since their replacement by Phe reduced the toxicity against Manduca sexta. The presence of hydrophobic residue is important at positions 117, 219, 226, and 455 since replacement by Cys affected either the crystal formation or the insecticidal activity of the toxin in contrast to replacement by Phe in these positions. Additionally, some mutants in positions 219, 316, and 455 were also affected in binding to brush border membrane vesicles (BBMV). This is the first report that studies the role of Trp residues in the activity of Cry toxins.
authors.affiliation: Departamento de Microbiología Molecular, Instituto de Biotecnología UNAM, Apdo. Postal 510-3, Cuernavaca, 27510 Morelos, Mexico.
authors.forename: Cristopher
authors.initials: C
authors.lastname: Padilla
authors.forename: Liliana
authors.initials: L
authors.lastname: Pardo-López
authors.forename: Gustavo
authors.initials: G
authors.lastname: de la Riva
authors.forename: Isabel
authors.initials: I
authors.lastname: Gómez
authors.forename: Jorge
authors.initials: J
authors.lastname: Sánchez
authors.forename: Georgina
authors.initials: G
authors.lastname: Hernandez
authors.forename: Maria Eugenia
authors.initials: ME
authors.lastname: Nuñez
authors.forename: Marianne P
authors.initials: MP
authors.lastname: Carey
authors.forename: Donald H
authors.initials: DH
authors.lastname: Dean
authors.forename: Oscar
authors.initials: O
authors.lastname: Alzate
authors.forename: Mario
authors.initials: M
authors.lastname: Soberón
authors.forename: Alejandra
authors.initials: A
authors.lastname: Bravo
mesh_terms: D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D004731:Endotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D006460:Hemolysin Proteins; D018402:Manduca / Q000254:growth & development*; D008871:Microvilli / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010572:Pest Control, Biological; D017510:Protein Folding; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D014364:Tryptophan