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2050-01-01
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THE N-TERMINAL THIRD OF THE BINB SUBUNIT FROM THE BACILLUS SPHAERICUS BINARY TOXIN IS SUFFICIENT FOR ITS INTERACTION WITH MIDGUT RECEPTORS IN CULEX QUINQUEFASCIATUS
Animais
Toxinas Bacterianas / Química
Toxinas bacterianas / metabolismo
Ligação, competitiva
Membrana celular
Culex / metabolismo
Eletroforese em Gel de Poliacrilamida
Immunoblotting
Proteínas de Insetos / química
Proteínas de Insetos / metabolismo
Larva
Microvilosidades / metabolismo
Mutagênese, Sítio-Dirigida
Mutação
Ligação proteica
Subunidades Proteicas
Receptores da Superfície Celular / química
Receptores da Superfície Celular / metabolismo
Proteínas Recombinantes
Affilliation
Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.
Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Microbiologia. Recife, PE, Brasil.
Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.
Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Microbiologia. Recife, PE, Brasil.
Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.
Abstract
Heterodimeric binary (Bin) toxin, the major insecticidal protein from Bacillus sphaericus, acts on Culex quinquefasciatus larvae through specific binding to the midgut receptor Cqm1, a role mediated by its 448-amino-acid-long BinB subunit. The molecular basis for receptor recognition is not well understood and this study attempted to identify protein segments and amino acid motifs within BinB that are required for this event. First, N- and C-terminally truncated constructs were evaluated for their capacity to bind to native Cqm1 through pull-down assays. These showed that residues N33 to L158 of the subunit are required for Cqm1 binding. Nine different full-length mutants were then generated in which selected blocks of three amino acids were replaced by alanines. In new pull-down assays, two mutants, in which residues (85) IRF(87) and (147) FQF(149) were targeted, failed to bind the receptor. Competition binding assays confirmed the requirements for the N-terminal 158 residues, and the (147) FQF(149) epitope, for the mutant proteins to compete with native Bin toxin when binding to membrane fractions from the insect midgut. The data from this work rule out the involvement of C-terminal segments in receptor binding, highlighting the need for multiple elements within the protein's N-terminal third for it to occur.
DeCS
Sequência de AminoácidosAnimais
Toxinas Bacterianas / Química
Toxinas bacterianas / metabolismo
Ligação, competitiva
Membrana celular
Culex / metabolismo
Eletroforese em Gel de Poliacrilamida
Immunoblotting
Proteínas de Insetos / química
Proteínas de Insetos / metabolismo
Larva
Microvilosidades / metabolismo
Mutagênese, Sítio-Dirigida
Mutação
Ligação proteica
Subunidades Proteicas
Receptores da Superfície Celular / química
Receptores da Superfície Celular / metabolismo
Proteínas Recombinantes
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