Pardo-López Liliana, Gómez Isabel, Rausell Carolina, Sanchez Jorge, Soberón Mario, Bravo Alejandra
Instituto de Biotecnologia, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, Cuernavaca 62250, Morelos, Mexico.
Biochemistry. 2006 Aug 29;45(34):10329-36. doi: 10.1021/bi060297z.
The primary action of Cry toxins produced by Bacillus thuringiensis is to lyse midgut epithelial cells in their target insect by forming lytic pores. The toxin-receptor interaction is a complex process, involving multiple interactions with different receptor and carbohydrate molecules. It has been proposed that Cry1A toxins sequentially interact with a cadherin receptor, leading to the formation of a pre-pore oligomer structure, and that the oligomeric structure binds to glycosylphosphatidyl-inositol-anchored aminopeptidase-N (APN) receptor. The Cry1Ac toxin specifically recognizes the N-acetylgalactosamine (GalNAc) carbohydrate present in the APN receptor from Manduca sexta larvae. In this work, we show that the Cry1Ac pre-pore oligomer has a higher binding affinity with APN than the monomeric toxin. The effects of GalNAc binding on the toxin structure were studied in the monomeric Cry1Ac, in the soluble pre-pore oligomeric structure, and in its membrane inserted state by recording the fluorescence status of the tryptophan (W) residues. Our results indicate that the W residues of Cry1Ac have a different exposure to the solvent when compared with that of the closely related Cry1Ab toxin. GalNAc binding specifically affects the exposure of W545 in the pre-pore oligomer in contrast to the monomer where GalNAc binding did not affect the fluorescence of the toxin. These results indicate a subtle conformational change in the GalNAc binding pocket in the pre-pore oligomer that could explain the increased binding affinity of the Cry1Ac pre-pore to APN. Although our analysis did not reveal major structural changes in the pore-forming domain I upon GalNAc binding, it showed that sugar interaction enhanced membrane insertion of soluble pre-pore oligomeric structure. Therefore, the data presented here permits to propose a model in which the interaction of Cry1Ac pre-pore oligomer with APN receptor facilitates membrane insertion and pore formation.
苏云金芽孢杆菌产生的Cry毒素的主要作用是通过形成裂解孔来裂解其目标昆虫的中肠上皮细胞。毒素与受体的相互作用是一个复杂的过程，涉及与不同受体和碳水化合物分子的多种相互作用。有人提出，Cry1A毒素依次与钙黏蛋白受体相互作用，导致形成孔前寡聚体结构，并且该寡聚体结构与糖基磷脂酰肌醇锚定的氨肽酶-N（APN）受体结合。Cry1Ac毒素特异性识别烟草天蛾幼虫APN受体中存在的N-乙酰半乳糖胺（GalNAc）碳水化合物。在这项工作中，我们表明Cry1Ac孔前寡聚体与APN的结合亲和力高于单体毒素。通过记录色氨酸（W）残基的荧光状态，研究了GalNAc结合对单体Cry1Ac、可溶性孔前寡聚体结构及其膜插入状态下毒素结构的影响。我们的结果表明，与密切相关的Cry1Ab毒素相比，Cry1Ac的W残基对溶剂的暴露情况不同。与单体不同，GalNAc结合特异性影响孔前寡聚体中W545的暴露，在单体中GalNAc结合不影响毒素的荧光。这些结果表明孔前寡聚体中GalNAc结合口袋存在细微的构象变化，这可以解释Cry1Ac孔前体与APN结合亲和力的增加。尽管我们的分析没有揭示GalNAc结合后成孔结构域I的主要结构变化，但它表明糖相互作用增强了可溶性孔前寡聚体结构的膜插入。因此，本文提供的数据允许提出一个模型，其中Cry1Ac孔前寡聚体与APN受体的相互作用促进膜插入和孔形成。
