Studies On The Structure And Functions Of Cry1Ac Protein From Bacillus Thuringiensis By Site-directed Mutagenesis

Bacillus thuringiensis(Bt)is the most used insecticidal microorganism that produces one or more insecticidal crystal(Cry)proteins deposited in the form of an intracellular parasporal crystal during sporulation,and it is a current hotpot to study the structure-function relationship of the Cry proteins. Cry1Ac protein,which is one of the widely researched Cry proteins with the strongest insecticidal activities against lepidoptera insects,was extensively used in pest control of agriculture,forestry and water ecology.But the structure-function relationship of Cry1Ac protein has not been well understood and need further discussed and explained with site-directed mutagenesis.In this paper,a uniqueβ18-β19 loop in the domainâ…¢of Cry1Ac5 was reported,and the Structural Significance of this loop in insecticidal activity of Cry1Ac5 was studied by site-directed mutagenesis.The major contents and results are as follows:The full-length cry1Ac5 gene was isolated and cloned from Bt 4.0718 strain,and the expression plasmid pHTAc35 was generated by cloning cry1Ac5 gene to shuttle vector pHT315.Acrystalliferous Bacillus thuringiensis cry^-B was transformed with pHTAc35 and well expressed a 130-kDa Cry1Ac5 protoxin,which offered the precondition for our latter research.A homology-based Cry1Ac model of Cry1Ac5 was constructed using SWISS-MODEL and Swiss-Pdb Viewer program,and its differences from Cry1Aa,Cry2Aa,Cry3Aa and Cry4Aa structures were analyzed.The Cry1Ac5 domainâ…¢has a distant relationship with other Cry1 proteins and its longβ18-β19 loop possesses a unique tryptophan(W)at residue 544 and a unique asparagine(N)at the loop apex(residue 546),as well as two consecutive serines(S)from residue 547 to 548.Alanine scanning mutations within the loop were initially generated and all mutants could produce bipyramidal crystals and express 130 kDa protoxins. When tested in toxicity,mutant N546A showed a slight enhanced mortality to Spodoptera exigua and Heliothis armigera,and mutant W544A exhibited a remarkable reduction in mortality to the two insects.After substantial analysis of Cry1Ac5 tertiary-structure,it can be found that both the residues N546 and S548,as well as the N543,are oriented towards the solvent,in the surface of the protein,probably indicating a putative role in interaction to other macromolecules.In contrast,side chain of residues W544,G545,S547 and I549 are in close contact to residues located in the large loop linkingβ21 andβ22,playing a conceivable structural role in local stability.Four further single mutants ofN546(N546G,N546D,N546K,N546Δ), three double mutants and a triple mutant were constructed,and toxins were purified by isoelectric precipitation and an AKTA purifier 100 system.When bioassayed against H.armigera using purified toxins,N546A(LC₅₀1.67μg/mL)showed 1.78 times increased toxicity than wide-type Cry1Ac(LC₅₀ 2.98μg/mL),and mutant N546D,N546K and N546Δshowed a great loss in toxicity.Toxin oligomerisation and proteolytic susceptibility assays revealed that this residue might not involved in toxin oligomerisation and maintaining the stability of toxin.Brush border membrane vesicles(BBMV)binding assay using biotinylated toxin revealed that the enhanced toxicity of mutant N546A was because of increased binding to BBMV,and reduction in toxicity of other mutants were caused by reduction in initial or inreversible binding to BBMV.The W544 was further conservatively substituted with phenylalanine(F) and tyrosine(Y)and nonconservatively replaced by cysteine(C).Bioassay and protein stability analysis indicated that the aromatic ring at this position was not absolutely necessary but the hydrophilic nature of the position had unfavorable influence to protein stability.Comparative analysis by SDS-PAGE showed that the protoxin of W544F was much more stable than the wild-type Cry1Ac,when treated with ultraviolet irradiation,trypsin and preserved at room temperature.The distance between two vertexes of the crystal of W544F was 0.6μm longer than that of the wild-type Cry1Ac under an atomic force microscope.Besides,the mutation W544F had similar insecticidal activity to wild-type Cry1Ac,but when treated with ultraviolet irradiation for 9 hours,it still maintained more than 4 times higher toxicity than the wild-type Cry1Ac,which might contribute to solving the major problem of field applications of Cry1Ac toxin.In this paper,the uniqueβ18-β19 loop in domainâ…¢of Cry1Ac5 toxin and its structural significance in the toxin functions was reported for the first time,which provided new biological evidences for domainâ…¢of Cry1Ac playing important roles in maintaining the stability of protein structure,and recognizing and binding with specific receptors.