Studies On The Structure And Function Of Cry1Ac Protein From Bacillus Thuringiensis And Its Interaction With 20-kb DNA

Bacillus thuringiensis(Bt) is the most widely used insecticidal microorganism that produces one or more insecticidal crystal(Cry) proteins deposited in the form of an parasporal crystal during sporulation, Cry1Ac,which is one of the deeply researched Cry proteins with the strongest insecticidal activities against lepidoptera insects,was extensively used in pest control of agriculture.But the structure-function relationship of Cry1Ac protein has not been well understood and need further investigated.It was reported that the parasporal crystals contained 20-kb heterofogous DNA fragments intimately associated with protoxin, which may play a key role in the assembling and stability of parasporal crystals as well as the activation of crystal toxin in the midgut of target larvae.However,the nature of the interaction of the Bt protoxin with DNA,the sequences and origin of 20kbDN remained to be investigated.The theoretical model of the three-dimensional structure of Cry1Ac was predicted by homology modeling on the structures of the Cry1Aa, and its differences from Cry1Aa,Cry2Aa,Cry3Aa and Cry4Aa structures were analyzed.It is composed of three structurally conserved domains. DomainⅠconsists of seven alpha helices in which helix 5 is surrounded by the others,forming a helical bundle.DomainⅡconsists of three antiparallel b-sheets joined in a Greek key topology,arranged in a b-prism,DomainⅢis formed by two antiparallel b-sheets forming aβ-sandwich in a jelly roll topology.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.20-kbDNA and the Cry1Ac5 were purified,and their interaction was analyzed using and fluorescence titration.The binding regions were in all the three domainsThe fluorescence titration curves of the calf thymus DNA and 20-kbDNA additions to the DNA-free Cry1A toxins were different.The binding of 20-kb was almost infinitely tight that was quite different from that of calf thymus DNA.Besides the structural features common to all DNA molecules,the toxins may recognizes specific nucleotide sequences feature present in the various 20-kb DNA fragments.It was confirmed that the mechanism of the fluorescence quenching of Cry1Ac5 induced by 20-kbDNA was static qunechnig.The binding constant K was 2.5×10~9,and the binding sites was 1.72.Molecular simulation using Cry1Ac5 and a piece of 20-bp DNA from 20-kb DNA showed that there were several binding sites on the three domains of Cry1Ac5.The binding site Ser283-Ala284-Gln285, Ser440-Ser441-Ser442 and Ser555-Leu556-Asn557 were deep in the grooves of the DNA to form the hydrophobic contacts.There were hydrogen bond interactions between the phosphorate backbone of DNA and three amino acids(Arg209,Asn212 and Gly339) of Cry1Ac5.The addition of 20-kb DNA to the activated Cry1Ac 5 solution in vitro reduced the degradation of the toxins under trypsin and ultraviolet irradiation that indicated the original existence of 20-kb DNA in toxins had the role of protection.In this paper,the mechanism of the interaction 20-kb DNA and Cry1Ac5 was reported for the first time,which provided new biological evidences for the interaction playing important roles in maintaining the stability of the toxin.