Structural And Functional Research On Bacillus Thuringiensis Cry51Aa1 Insecticidal Crystal Protein

Since the first structure of Cry3 Aa was solved in the year of 1991, structural biology of Bacillus thuringiensis(Bt) toxin has been developed rapidly, which promoted the development of insecticidal research field, and advanced the usage of Bt toxin in agriculture and forestry. With the purpose of exploring the structure of novel Bt toxin, explaining insecticidal mechanism, expanding Bt application range, and countering insects resistance in the field, this study takes a relatively thorough study on the structural and functional research of the newly discovered Cry51Aa1 toxin by using the X-ray crystallography techniques The results are summarized as follows:1. This study determined the full-length structure of Cry51Aa1 toxin with the specific toxicity towards Colorado Potato Beetle(CPB) larvae, and presented distinct threedimensional architecture from traditional three-domain type Cry toxin. It was the first report in China that a new relationship between the novel structure of Bt crystal protein and the insecticidal activity has been established. Moreover, the resolution record of Bt insecticidal toxins was renovated into 1.65 ?.2. The overall molecule is mainly composed of elongated β-strands and could be divided into three domains. Domain I is at the N-terminus of the structure and contains a majority of helices. Domain II locates at the center part, having a β-hairpin and a β-sheet. Domain III is from the C terminus and consists of two β-sheets. Domain II and domain III present as the ‘β-sandwich’ architecture.3. Further analysis reveals that Domain I has plenty of aromatic residues, which is suggested to be involved in membrane interaction and structural stability. Domain II is dominant with serine and threonine residues, and has a typical amphipathic antiparallel β-hairpin with a glycine-rich loop at the tip. Domain II is proposed to play a key role in oligomerization and membrane penetration. Domain III has an intact long C-terminal tail and is thought to be related to proteolytic digestion and conformational change.4. The molecules of Cry51Aa1 protoxin were found to assemble as monomers in asymmetric unit but as crystallographic dimers in crystals and solution. Structural analysis implies that, after trypsin digestion, monomer-monomer interactions would be broken, dimer splits into monomers, and monomers aggregate into hexamer, the latter of which could be the Cry51Aa1 oligomeric state when interacting with the membrane. Proteolytic digestion could also break the intramolecular hydrogen bonds at domainboundaries, then influences the stability of the amphipathic β-hairpin consequently. The glycine-rich loop may guide the released amphipathic β-hairpin to stretch out towards the membrane, and abundant Ser/Thr residues may help the amphipathic β-hairpins gather together to form a stable pre-pore oligomer.5. Cry51Aa1 could be classified into aerolysin-type β-pore-forming toxins(β-PFTs) family, based on the distribution similarities of helices and β-strands, homology of the three-dimensional structure, as well as the typical amphipathic β-hairpin. The Cry51Aa1 monomer structure is also compared and analyzed to the family members of the aerolysin-type β-PFTs.6. Insect bioassay experiment demonstrates that Cry51Aa1 has relatively high insecticidal toxicity against CPB larvae. Functional testing on coleopteran insects verifies the insecticidal spectrum of Cry51 A toxin group, which makes this group as a Coleopteranspecific category with novel structure. Meanwhile, the Anopheles gambiae Ag55 cell line that are treated with activated Cry51Aa1 presents morphologic changes with the evidence of cellular swelling, and it is speculated that the toxin could influence the integrity of cell membrane, that is, the activated Cry51Aa1 has the cytotoxicity activity towards the Ag55 cell ine.By using the structural biology methods, this study explores and summarizes an integrated technique pipeline that is suitable for the purification, crystallization, X-ray diffraction, and structure determination of Bt insecticidal crystal protein expression system. The structure of Cry51Aa1 insecticidal toxin joins as a new study object of the Bt structural research field, and also provides unique research ideas. The functional studies reveal and broaden the toxic range of Cry51Aa1. Moreover, the functional evidences not only confirms that there is a certain kind of interaction between Cry51 A toxin group and coleopteran or hemipteran insects membrane, but implie this kind of interaction on the insect membrane remains similar to the aerolysin-type β-PFTs. In-depth study and analysis on Cry51Aa1 insecticidal crystal structure may contribute to further explanation on the relationship of Bt structure and function, benefit the research on insecticidal working mechanism, and also offer basic reference to the insect control in the future.Before the statement of the structural and functional research on Cry51Aa1, this study also provides a detailed review on current structural and functional research progress of Bt insecticidal toxin. The review mainly focuses on the analysis, classification and conclusion of different characteristics of three categories of Bt toxin. Similarities and dissimilarities ofhomologous structures and corresponding functions are compared and described. Also, the working mechanisms of insecticidal Bt toxin are discussed, which could provide some ideas and experience for the following research on Cry51Aa1 toxin. Additionally, as to highlight the research features of modern structural biology, this study also gives a brief summary on the concept and method of protein crystallography.