Molecular Basis for Bacillus thuringiensis Cry1Ab Toxin Specificity: Two Structural Determinants in the Manduca sexta Bt-R1 Receptor Interact with Loops 详细信息    查看全文

• 作者：Isabel G&oacute ; mez ; Donald H. Dean ; Alejandra Bravo ; and Mario Sober&oacute ; n
• 刊名：Biochemistry
• 出版年：2003
• 出版时间：September 9, 2003
• 年：2003
• 卷：42
• 期：35
• 页码：10482 - 10489
• 全文大小：118K
• 年卷期：v.42,no.35(September 9, 2003)
• ISSN：1520-4995

文摘

The identification of epitopes involved in Cry toxin-receptor interaction could provide insightsinto the molecular basis of insect specificity and for designing new toxins to overcome the potentialproblem of insect resistance. In previous works, we determined that the Manduca sexta Cry1A cadherin-like receptor (Bt-R₁) interacts with Cry1A toxins through epitope ⁸⁶⁵NITIHITDTNN⁸⁷⁵ and by loop 2 ofdomain II in the toxin (Gomez, I., Miranda-Rios, J., Rudiño-Piñera, E., Oltean, D. I., Gill, S. S., Bravo,A., and Soberón, M. (2002) J. Biol. Chem. 277, 30137-30143.). In this work, we narrowed to 12 aminoacids a previously identified Bt-R₁ 66 amino acids epitope (Dorsch, J. A., Candas, M., Griko, N. B.,Maaty, W. S. A., Midbo, E. G., Vadlamudi, R. K., and Bulla, L. A., Jr. (2002) Insect Biochem. Mol. Biol.32, 1025-1036) and identified loop -8 of Cry1Ab domain II as its cognate binding epitope. Two aminoacid Bt-R₁ toxin binding regions of 70 residues, one comprised of residues 831-900 containing the ⁸⁶⁵NITIHITDTNN⁸⁷⁵ epitope (TBR1) and the other comprised of residues 1291-1360 (TBR2) were clonedby RT-PCR and produced in Escherichia coli. Cry1A toxins bind with the two TBR regions in contrastwith the nontoxic Cry3A toxin. The loop 2 synthetic peptide competed with the binding of Cry1Ab toxinto both TBR regions in contrast to the -8 synthetic peptide that only competed with Cry1Ab binding toTBR2. Western blots and competition ELISA analysis showed that the Cry1Ab loop 2 RR368-9EE mutantdid not show observable binding to TBR1 but still bound the TBR2 peptide. This result suggests thatloop -8 interacts with the TBR2 region. Competition ELISA analysis of Cry1Ab binding to the twoTBR peptides revealed that the toxin binds the TBR1 region with 6-fold higher affinity than the TBR2region. The amino acid sequence of TBR2 involved on Cry1Ab interaction was narrowed to 12 aminoacids, ¹³³¹IPLPASILTVTV¹³⁴², by using synthetic peptides as competitors for Cry1Ab binding to Bt-R₁.Our results show that the specificity of Cry1A involves at least two structural determinants on bothmolecules.