title: Mechanistic insights into the first Lygus-active β-pore forming protein.
abstract: The cotton pests Lygus hesperus and Lygus lineolaris can be controlled by expressing Cry51Aa2.834_16 in cotton. Insecticidal activity of pore-forming proteins is generally associated with damage to the midgut epithelium due to pores, and their biological specificity results from a set of key determinants including proteolytic activation and receptor binding. We conducted mechanistic studies to gain insight into how the first Lygus-active β-pore forming protein variant functions. Biophysical characterization revealed that the full-length Cry51Aa2.834_16 was a stable dimer in solution, and when exposed to Lygus saliva or to trypsin, the protein underwent proteolytic cleavage at the C-terminus of each of the subunits, resulting in dissociation of the dimer to two separate monomers. The monomer showed tight binding to a specific protein in Lygus brush border membranes, and also formed a membrane-associated oligomeric complex both in vitro and in vivo. Chemically cross-linking the β-hairpin to the Cry51Aa2.834_16 body rendered the protein inactive, but still competent to compete for binding sites with the native protein in vivo. Our study suggests that disassociation of the Cry51Aa2.834_16 dimer into monomeric units with unoccupied head-region and sterically unhindered β-hairpin is required for brush border membrane binding, oligomerization, and the subsequent steps leading to insect mortality.
authors.affiliation: Monsanto Company, Chesterfield, MO, USA. Electronic address: agoston.jerga@monsanto.com.
authors.forename: Agoston
authors.initials: A
authors.lastname: Jerga
authors.affiliation: Monsanto Company, Chesterfield, MO, USA.
authors.forename: Danqi
authors.initials: D
authors.lastname: Chen
authors.affiliation: Monsanto Company, Chesterfield, MO, USA.
authors.forename: Chunfen
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Entomology, Kansas State University, Manhattan, KS, USA.
authors.forename: Jinping
authors.initials: J
authors.lastname: Fu
authors.affiliation: Monsanto Company, Chesterfield, MO, USA.
authors.forename: Jean-Louis K
authors.initials: JK
authors.lastname: Kouadio
authors.affiliation: Monsanto Company, Chesterfield, MO, USA.
authors.forename: Yanfei
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Monsanto Company, Chesterfield, MO, USA.
authors.forename: Stephen M G
authors.initials: SMG
authors.lastname: Duff
authors.affiliation: Monsanto Company, Chesterfield, MO, USA.
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Howard
authors.affiliation: Monsanto Company, Chesterfield, MO, USA.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Rydel
authors.affiliation: Monsanto Company, Chesterfield, MO, USA.
authors.forename: Artem G
authors.initials: AG
authors.lastname: Evdokimov
authors.affiliation: Monsanto Company, Chesterfield, MO, USA.
authors.forename: Parthasarathy
authors.initials: P
authors.lastname: Ramaseshadri
authors.affiliation: Monsanto Company, Creve-Coeur, MO, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Evans
authors.affiliation: Monsanto Company, Chesterfield, MO, USA.
authors.forename: Renata
authors.initials: R
authors.lastname: Bolognesi
authors.affiliation: Department of Entomology, Kansas State University, Manhattan, KS, USA.
authors.forename: Yoonseong
authors.initials: Y
authors.lastname: Park
authors.affiliation: Monsanto Company, Chesterfield, MO, USA.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Haas
mesh_terms: D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000633:toxicity / Q000648:ultrastructure*; D001665:Binding Sites; D004731:Endotoxins / Q000737:chemistry* / Q000633:toxicity; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000633:toxicity / Q000648:ultrastructure*; D020062:Heteroptera / Q000737:chemistry*; D019476:Insect Proteins; D052899:Pore Forming Cytotoxic Proteins / Q000737:chemistry* / Q000633:toxicity / Q000648:ultrastructure*; D011485:Protein Binding; D011487:Protein Conformation; D012463:Saliva / Q000737:chemistry*; D013534:Survival; D014357:Trypsin / Q000737:chemistry
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D019476:Insect Proteins; D052899:Pore Forming Cytotoxic Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D014357:Trypsin
keywords: Bt cotton; Cry51Aa2; Lygus; Pore-forming protein; Protease activation; Toxin binding