title: Hydropathic complementarity determines interaction of epitope (869)HITDTNNK(876) in Manduca sexta Bt-R(1) receptor with loop 2 of domain II of Bacillus thuringiensis Cry1A toxins.
abstract: In susceptible insects, Cry toxin specificity correlates with receptor recognition. In previous work, we characterized an scFv antibody (scFv73) that inhibits binding of Cry1A toxins to cadherin-like receptor. The CDR3 region of scFv73 shared homology with an 8-amino acid epitope ((869)HITDTNNK(876)) of the Manduca sexta cadherin-like receptor Bt-R(1) (Gomez, I., Oltean, D. I., Gill, S. S., Bravo, A., and Soberón, M. (2001) J. Biol. Chem. 276, 28906-28912). In this work, we show that the previous sequence of scFv73 CDR3 region was obtained from the noncoding DNA strand. However, most importantly, both scFv73 CDR3 amino acid sequences of the coding and noncoding DNA strands have similar binding capabilities to Cry1Ab toxin as Bt-R(1) (869)HITDTNNK(876) epitope, as demonstrated by the competition of scFv73 with binding to Cry1Ab with synthetic peptides with amino acid sequences corresponding to these regions. Using synthetic peptides corresponding to three exposed loop regions of domain II of Cry1Aa and Cry1Ab toxins, we found that loop 2 synthetic peptide competed with binding of scFv73 to Cry1A toxins in Western blot experiments. Also, loop 2 mutations that affect toxicity of Cry1Ab toxin are affected in scFv73 binding. Toxin overlay assays of Cry1A toxins to M. sexta brush border membrane proteins showed that loop 2 synthetic peptides competed with binding of Cry1A toxins to cadherin-like Bt-R(1) receptor. These experiments identified loop 2 in domain II of as the cognate binding partner of Bt-R(1) (869)HITDTNNK(876). Finally, 10 amino acids from beta-6-loop 2 region of Cry1Ab toxin ((363)SSTLYRRPFNI(373)) showed hydropathic pattern complementarity to a 10-amino acid region of Bt-R(1) ((865)NITIHITDTNN(875)), suggesting that binding of Cry1A toxins to Bt-R(1) is determined by hydropathic complementarity and that the binding epitope of Bt-R(1) may be larger than the one identified by amino acid sequence similarity to scFv73.
authors.affiliation: Departamento de Microbiologia Molecular, Instituto de Biotecnologia, Universidad Nacional Autónoma de México, Apdo postal 510-3, Cuernavaca, Morelos 62250, México.
authors.forename: Isabel
authors.initials: I
authors.lastname: Gomez
authors.forename: Juan
authors.initials: J
authors.lastname: Miranda-Rios
authors.forename: Enrique
authors.initials: E
authors.lastname: Rudiño-Piñera
authors.forename: Daniela I
authors.initials: DI
authors.lastname: Oltean
authors.forename: Sarjeet S
authors.initials: SS
authors.lastname: Gill
authors.forename: Alejandra
authors.initials: A
authors.lastname: Bravo
authors.forename: Mario
authors.initials: M
authors.lastname: Soberón
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D015374:Biosensing Techniques; D017931:DNA Primers; D000939:Epitopes / Q000737:chemistry*; D018402:Manduca; D008958:Models, Molecular
chemical_list: D001427:Bacterial Toxins; D017931:DNA Primers; D000939:Epitopes