The Study Of Structure-function Relationship Of Cry1Ab Protein And Cry1Ab1-binding Nonreceptor Proteins From Plutella Xylostella

Bacillus thuringiensis(Bt) is an insecticidal bacterium that has been applied to pest control worldwide. Its insecticidal activity mainly comes from the crystal proteins(Cry) produced during sporulation. The lack of efficient Cry proteins that target several important pests and insect resistance agaisnt Cry are two major threats to the long-term application of Cry proteins. In this study, we investigated the structure- function relationship of Cry1Ab1 and identified non- receptor Cry1Ab1-binding proteins from the midgut juice of Plutella xylostella. Results are shown as follows:(1) The 3-D structure of the toxic core for all available 34 Cry1 Ab proteins were constructed by the method of homology modeling. Based on the secondary structure pattern, four different groups were identified and named as Cry1AbⅠ, Cry1AbⅡ,Cry1AbⅢ, and Cry1AbⅣ. The three Cry1 Ab proteins, Cry1Ab2, Cry1Ab7 and Cry1Ab28 were recognized as Cry1AbⅡ, Cry1AbⅢ, and Cry1AbⅣ, respectively.The other 31 Cry1 Ab proteins were grouped as Cry1AbⅠ, and were further divided into three subgroups based on 3-D structural differences, Cry1AbⅠ3(Cry1Ab33only), Cry1AbⅠ2(Cry1Ab31 only), and Cry1AbⅠ1(the rest of Cry1AbⅠ). The structural differences among different Cry1 Ab groups and subgroups were presented in details. Sequence alignment revealed four different residues existed between Cry1Ab1(Cry1AbⅠ) and Cry1Ab7(Cry1AbⅢ), including: A450 P, F537 L, P545 I and T568 I. Structure comparison showed that residues 447-449 form β-sheet in Cry1Ab1 while a loop in Cry1Ab7, and this resulted from residue 450. The residue450 is located in loop3 of the DomainⅡ, which might involve in Cry-receptor binding. Other residues(537, 545 and 568) are located in the β17, β18 and β20 of DomainⅢ, respectively, which might involve in Cry-receptor binding or structural stability.(2) The structure- function relationship between Cry1Ab1 and Cry1Ab7 was investigated by site-directed mutagenesis and bioassay. Based on structure analysis,the residue 450 of Cry1Ab1 was mutagenized. Cry1Ab1 showed 61.2% and 78.9%corrected mortality at the concentration of 1 μg/g and 128 μg/g, respectively against P.xylostella, while mutant A450 P 45.6% and 58.9%, indicated DomainⅡ loop3 played an important role in its activity against P. xylostella. Additionally, mutant A450 G showed 34.5% and 55.6% corrected mortality, indicated the side chain of residue 450 was involved in insecticidal activity. However, Mutant A450 P had no toxicity against Aedes aegypti at the concentration of 50 μg/m L, which indicated that the loop3 structure was not enough to determine the activity of Cry1Ab7 against A.aegypti.(3) Molecular docking between Cry and receptor were performed to explain the different insecticidal activities between Cry1Ab1 and Cry1Ab7. The different structure was found to be involved in the binding with P. xylostella APN, this might result in lower insecticidal activity of mutant A450 P compared to Cry1Ab1. The different structure between Cry1Ab1 and Cry1Ab7, and Domain II of Cry1Ab1 were found not to be involved in the binding with A.aegypti receptors APN and ALP, which were believed to be the reasons why Cry1Ab1 its mutant A450 P had no activities against A. aegypti.(4) The nonreceptor Cry-binding proteins were first identified from midgut juice of P. xylostella. Five putative Cry1Ab-binding proteins were selected for identification by the LC-MS/MS, they were tubulin, glutathione synthetase, ATP synthase, arginine kinase, and trypsin.(5) The nonreceptor Cry-binding proteins of P. Xylostella midgut sampled at different time after fed with Cry1Ab1 were compared. It was found that the amounts of two proteins, translation elongation factor 1-α and cytochrome c oxidase, were gradually decreased then dissappeared, which indicated the two proteins were involved in the insecticidal process.The results presented here provided new insights into structure-function relationship of Cry proteins. The results suggested that there existed various Cry-binding non-receptor proteins in insect midgut, which may interfere with Cry toxin binding to the receptors and thus impair toxin specificity and insecticidal activity, which may also interfere with some specific pathways in insects to facilitate the insecticidal process.