NMR Structure And Promoter Activity Analysis Of The Sterol Carrier Protein 2 From Helicoverpa Armigera

The cotton bollworm, Helicoverpa armigera (Hubner) (Lepidoptera:Noctuidae) causes serious crop damage every year all over the world and poses a great threat to the economics of global agricultural production. It has developed strong resistance to many insecticides and transgenic Bt cotton. There is an urgent need to seek safer insecticides with new modes of action to effectively control the cotton bollworm. Sterol Carrier Protein-2 (SCP-2) is an important non-specific lipid transfer protein in insects and appears to be a potential new insecticide target.In order to elucidate the structure and function of Helicoverpa armigera SCP-2 (HaSCP-2), NMR spectroscopy, docking simulations, mutagenesis and bioassays were performed. The main results in this study are as follows:(1) pGEX-KG-HaSCP-2 recombinant expression vector was constructed and induced with final concentration of 0.2mM IPTG at 28 °C for 10 hours, Then HaSCP-2 protein was purified by GST affinity chromatography, thrombin digestion, anion exchange chromatography and gel filtration column separation. (2) The HaSCP-2 protein were truncated and optimized based on the reports, protein sequences alignment, MALDI-TOF mass spectrometry and NMR HSQC spectra analysis. Finally,16 amino acids in N terminal and 4 amino acids in C terminal were removed, TritonX-100 molecule was also used to improve the NMR spectroscopy. There was no affinity difference between the deduced SCP-2 and truncated SCP-2 proteins with substrate by NBD-cholesterol binding assay. (3) It was showed that HaSCP-2 was composed of five a-helices and four stranded β-sheets. The folds of a-helices and β-sheets interacted together to form a hydrophobic cavity with putative entrance and exit openings, which served as a tunnel for accommodating and transporting of lipids. The proteins with structures similar to HaSCP-2 had been analysis, and the most similar structure is that of Oryctolagus cuniculus (European rabbit) with sequence identify of 47%. The atomic coordinates of the HaSCP-2 has been deposited in the Protein Data Bank with the accession code of 4UEI. (4) Flexible simulation docking analysis by SYBYL-7.3 software showed that sterols could interact with HaSCP-2 via important hydrophobic sites, such as F53, F89,191, F10, 1117, Q131, which could be potential targets for insecticides. Mutagenesis experiments indicated Y51A, Y51W, F53A, F53W, F89A, F110W, I91M, I117M, Q131A mutation protein decreased the affinity with substrate of cholesterol. (5) HaSCP-2 showed high cholesterol binding activity and SCP-2 inhibitors (SCPIs) could inhibit the biological activity of HaSCP-2 by molecular docking analysis and competitive binding assay. SCPI-treated larvae at young stage showed a significant decrease of cholesterol uptake in vivo.On the other hand, the promoter sequences of HaSCP-X/SCP-2 gene were cloned and analyzed.The main results of this study are as follows:(1) An about -8kb sequence of the HaSCP-X/SCP-2 promoter was successfully cloned by chromosome walking. (2) A series of deleted promoter/report gene constructs were build, and the transcription activity of the promoter was studied by using dual luciferase reporter assays. It was found that-1.8kb promoter sequence showed the highest activity, which suggested that-1.8kb sequence is important for the HaSCP-X/SCP-2 promoter. (3)The experiment of 5’-RACE demonstrated that there are multiple weak start sites that are distributed over a region of about 100 nt in HaSCP-X/SCP-2 promoter. (4) The bioinformatics analysis of HaSCP-X/SCP-2 promoter predicted that some putative cis-regulation elements such as CEBP, AP-1, TGIF motifs upstream of the first initiator were maybe responsible for the regulation of HaSCP-X/SCP-2 gene expression.Our study have described three-dimensional structure of HaSCP-2 by NMR for the first time, the important functional sites of the protein were revealed, and the promoter sequences which involved in regulation of HaSCP-2 expression was cloned and analyzed. All these studies will facilitates the screening of effective insecticides targeting the insect cholesterol metabolism.