The Classification And Structure-function Relationship Of Cry8 Proteins Based On 3-D Structure Differences

Bacillus thuringiensis(Bt)is widely used insects pests all over the world.Its insecticidal activity is mainly due to the insecticidal crystal proteins(Cry).Cry8 proteins toxic to Scarabaeidat,underground pests.So far,53 Cry8 proteins have been discovered.Resistance development is one of the two major constraints on the long-term application of Bt and Cry proteins,while the other one is lack of available Cry proteins with insecticidal effect on important pests.This paper studies the classification and structure-function relationship of Cry8 proteins based on 3-D structure differences and the binding of Cry8 proteins to their receptors.The main results are as follows:The 3-D structure of the toxic core for available 53 Cry8 proteins were constructed by the method of homology modeling.Based on the the secondary structure pattern,thirty-four different groups were identified and named as Cry8A?-?,Cry8B?-?,Cry8C?-?,Cry8D?-?,Cry8E?,Cry8F?-?,Cry8G?-?,Cry8H?,Cry8J?,Cry8I?-?,Cry8K?-?,Cry8L?,Cry8M?,Cry8N?,Cry8P?-?,Cry8 Q ?,Cry8R?and Cry8T?.Based on the above classification,all the members in the same group were subdivided according to the degree of overlapping in their 3-D Structure.Cry8 E ?was subdivided into two subtype: Cry8 E ? Class1(Cry8Ea2,Cry8Ea4 and Cry8Ea5),Cry8E?Class2(Cry8Ea1).Similarly,each in turm Cry8 F ?was subdivided into Cry8F?Class1(Cry8Fa2)and Cry8F?Class2(Cry8Fa3),Cry8I?was subdivided into Cry8I?Class1(Cry8Ia1),Cry8I?Class2(Cry8Ia2)and Cry8 I?Class3(Cry8Ia3),Cry8M?was subdivided into Cry8M?Class1(Cry8Ma1,Cry8Ma3)and Cry8 M ?Class2(Cry8Ma2).3-D structural difference in Cry8-type proteins were thoroughly analyzed,then their relationships between structure and insecticidal activity were discussed by selecting several groups of Cry8 proteins based on the present data of insecticidal activity.Cry8 proteins in some groups have the same insecticidal activity against the same pest,while in other groups,their members' s insecticidal activity varies.Results are as follows: 1.Cry8Bb1 and Cry8Bc1 were active against Leptinotarsa Decemlineata,Diabrotica undecimpunctata and Diabrotica virgifera.Both have the same conformation of Loop-?8.2.Cry8Ca1 and Cry8Ca2 were active against Anomala cuprea.Both have the same conformation of Loop-?8.3.Cry8Da1 and Cry8Db1 had toxicity against both adults and larvae of the Japanese beetle,but Cry8Da1 is slightly more toxic than Cry8Db1.They have the same conformation of Loop-?8,while Loop?3-?4 structures are different between these two proteins.We assumed that the reason why they have different structures is that N23 S and Y44 C.4.Cry8Ea1 and Cry8Ea2 were active against Holotrichia oblita(H.oblita)and Holotrichia parallela(H.parallela).Both have the same conformation of Loop-?8,Loop1 and Loop3.5.Cry8Fa1 was active against H.oblita and H.parallela,but Cry8Fa2 was not active against either H.oblita or H.parallela.Structural differences were found in their Loops.We assumed that this difference may lie in different structures of D448 N.6.Cry8Ga1 was active against H.oblita,while Cry8Ga2 was not.We assumed that the different conformations in their Loop3 are caused by the difference in V337 M.7.Cry8Ea1 and Cry8Ga1 were active against H.oblita,H.parallela and Anomala cuprea,while they were not active against Plutella xylostella,Helicoverpa armigera,Tenebrio molitor or Tribolium castaneum.All have the same conformation of Loop-?8 and Loop3.The intercomparison above suggests that the difference in Loop has important role in the binding of proteins and its receptors,especially conformational differences in Loop-?8.Loop?3-?4 may have an impact on the strength of insecticidal activity.These facts are evidence that the new classification system established in this study is reasonable.Protein-protein docking of every two among Cry8Ea1 protein,APN and ?-Tubulin showed that ten overlapping sites of APN and ?-Tubulin are located on Cry8Ea1's Domain ?.Most of them were located at the site of 267 E,464-465 AN,477S,479 N,573-577 VGGSV.This docking result may lead us to deduce that ?-Tubulin plays a role in the H.parallela resistance to toxin by preventing Cry8Ea1 from binding with APN of H.parallela.Protein-protein docking of every two among Cry8Ea1 protein,lection and APN showed that Cry8Ea1 can dock with the other two proteins successfully,no overlapping site existing in the process.The result suggests that lection has no influence on the insecticidal activity of Cry8Ea1 because there is no possible competition between H.parallela lection and APN.At last,Loop-?8 is involved in Cry8 Ea protein-Membrane alanyl aminopeptidase of H.oblita interactions,and both located areas are 273-275 GKH,which is in agreement with the intercomparison result that Loop-?8 in the two proteins share the same conformation.The discovery was concluded that Loop-?8 plays an important role in the specific binding between Cry8 Ea protein-Membrane alanyl aminopeptidase.Loop?4-?5 is involved in APN's binding with Cry8Ea1,Cry8Ea2 respectively,and both located areas are125-129 VTGYE,which is the same as the result of structural intercomparison between two proteins.This discovery was concluded that Loop?4-?5 plays an important role in the pore formation of the larvae of H.parallela.All the analysis above shows that this research is based on the new classification method of 3-D structure in active zone.This classification method is more propitious to explore the relationship between 3-D structure and insecticidal activity than the classification method on the basis of insecticidal spectrum and amino acid sequence homology.The research explores the relationship between structure and function based on the 3-D structural difference as well as the establishment of a new relationship between new protein structure in Bacillus thuringiensis and insecticidal activity.Molecular Docking technique was used for exploring the major connecting sites of amino acid in docking area of receptors and Cry toxin in this paper.Its conclusions not only provide the theoretical basis for kinds of mutant designs and clone in the future,but also have great significance in expanding insecticide spectrum of Cry8 parasporal crystal protein,improving Insecticidal toxicity and delaying the onset of insecticidal resistance.