Pacheco Sabino, Gómez Isabel, Sánchez Jorge, García-Gómez Blanca-Ines, Soberón Mario, Bravo Alejandra
Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico.
Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico
Appl Environ Microbiol. 2017 Sep 29;83(20). doi: 10.1128/AEM.01515-17. Print 2017 Oct 15.
three-domain Cry toxins kill insects by forming pores in the apical membrane of larval midgut cells. Oligomerization of the toxin is an important step for pore formation. Domain I helix α-3 participates in toxin oligomerization. Here we identify an intramolecular salt bridge within helix α-3 of Cry4Ba (D111-K115) that is conserved in many members of the family of three-domain Cry toxins. Single point mutations such as D111K or K115D resulted in proteins severely affected in toxicity. These mutants were also altered in oligomerization, and the mutant K115D was more sensitive to protease digestion. The double point mutant with reversed charges, D111K-K115D, recovered both oligomerization and toxicity, suggesting that this salt bridge is highly important for conservation of the structure of helix α-3 and necessary to promote the correct oligomerization of the toxin. Domain I has been shown to be involved in oligomerization through helix α-3 in different Cry toxins, and mutations affecting oligomerization also elicit changes in toxicity. The three-dimensional structure of the Cry4Ba toxin reveals an intramolecular salt bridge in helix α-3 of domain I. Mutations that disrupt this salt bridge resulted in changes in Cry4Ba oligomerization and toxicity, while a double point reciprocal mutation that restored the salt bridge resulted in recovery of toxin oligomerization and toxicity. These data highlight the role of oligomer formation as a key step in Cry4Ba toxicity.
三结构域Cry毒素通过在幼虫中肠细胞顶端膜上形成孔道来杀死昆虫。毒素的寡聚化是形成孔道的重要步骤。结构域I的α-3螺旋参与毒素的寡聚化。在此，我们鉴定出Cry4Ba的α-3螺旋内的一个分子内盐桥（D111-K115），该盐桥在三结构域Cry毒素家族的许多成员中是保守的。单点突变如D111K或K115D导致蛋白质毒性严重受影响。这些突变体在寡聚化方面也发生了改变，并且突变体K115D对蛋白酶消化更敏感。电荷反转的双点突变体D111K-K115D恢复了寡聚化和毒性，表明该盐桥对于α-3螺旋结构的保守非常重要，并且是促进毒素正确寡聚化所必需的。已表明在不同的Cry毒素中，结构域I通过α-3螺旋参与寡聚化，影响寡聚化的突变也会引起毒性变化。Cry4Ba毒素的三维结构揭示了结构域I的α-3螺旋中的一个分子内盐桥。破坏该盐桥的突变导致Cry4Ba寡聚化和毒性发生变化，而恢复盐桥的双点反向突变导致毒素寡聚化和毒性恢复。这些数据突出了寡聚体形成作为Cry4Ba毒性关键步骤的作用。
