Solution Structure Characteristics Of Anti-asthma Target Transgelin-2 Protein And Binding Sites Of Its Agonists Based On NMR Technique

Objective:The previous work of our research group found that Transgelin-2 protein was a key target protein of acupuncture in the anti-asthma treatment,by using genomics,proteomics,bioinformatics and other theories and technologies.However,the lack of full-length structure of Transgelin-2 protein is not conducive to the accurate identification of its biological function,and the study of the biological mechanism of acupuncture against asthma has brought uncertainty.Therefore,in order to study the structural characteristics of the protein,the Transgelin-2 protein labeled with high purity stable isotope was obtained through the prokaryotic expression system in this study.The full-length solution structure of transgelin-2 protein was characterized by the three-dimensional Nuclear Magnetic Resonance(NMR)technique.Basis on that,the binding sites of Compound of Acupuncture Anti-Asthmatic Target(CAT),which specifically binds to Transgelin-2 protein and has the function of relaxing tracheal smooth muscle,were identified.To provide scientific basis for further elucidating the molecular mechanism of acupuncture against asthma.Method:1.The primary structure of Transgelin-2 protein obtained in the previous class group was analyzed.The physical and chemical properties and natural disordered regions of Transgelin-2 protein were analyzed by using the software of ExPASY and PONDR online analysis method,respectively,to provide theoretical basis and direction for the selection of transgelin-2 protein structure characterization methods.2.The prokaryotic expression vector of pCold Ⅱ-Transgelin-2 was constructed,and expression by E.coli expression system,and the recombinant Transgelin-2 protein protein labeled with non-labeled,15N-labeled and 15N-13C double labeled stable isotopes were induced and expressed.Then the recombinant Transgelin-2 protein protein were purified by Ni2+affinity chromatography,cation exchange chromatography and gel filtration chromatography successively.The recombinant Transgelin-2 protein with high purity,high concentration and uniformity was obtained to provide tool protein for subsequent experiments.3.NMR technology was used to characterize the solution structure of the full-length Transgelin-2 protein.NMR analysis software SPARKY was used to identify the atomic chemical shift of the main chain of Transgelin-2 protein,and the secondary structure of the protein was calculated by using NMRPipe software Talos-N program.This will lay a foundation for the study of molecular mechanism of action.4.The binding strength of Transgelin-2 protein with different agonist small molecule compounds CAT was detected by Surface Plasmon Resonance(SPR)and Isothermal Titration Calorimetry(ITC),and the speed of binding and dissociation and the stability of the complex were analyzed.5.Furthermore,NMR titration experiments were used to examined the interaction binding sites between Transgelin-2 protein and the binding small molecule compound CAT-180,and analyzed the binding interface between Transgelin-2 protein and CAT-180.Results:1.Transgelin-2 protein was predicted to be a partially intrinsically disordered proteins with a continuous disordered structure of 52 amino acid residues at its C-terminal.2.The prokaryotic expression vector of pCold II-Transgelin-2 was successfully constructed.The expression of unlabeled recombinant Transgelin-2 protein was successfully induced,and the recombinant protein with the concentration of 10.5 mg/mL was obtained by purification step by step.By optimizing the M9 medium,Transgelin-2 protein with 15N labeling and 15N-13C double labeling was successfully expressed,and the recombinant protein with 3.4 mg/mL concentration was obtained by purification step by step.3.The purified recombinant Transgelin-2 protein of high purity and high concentration of acupuncture anti-asthma target was replaced into the buffer solution required for NMR experiment,and the stability of Transgelin-2 protein was determined to be 7 days.4.Two-dimensional 1H-15N HSQC spectra and three-dimensional HNCO,HN(CA)CO,HNCA,HN(CO)CA,HNCACB and CBCA(CO)NH spectra of Transgelin-2 protein solution were collected to complete the identification of hydrogen,carbon and nitrogen signals of 92%non-proline residues(185/201).5.According to the high-resolution NMR results of the Transgelin-2 protein,the chemical shift of the main chain was obtained and the secondary structure of the protein was calculated.The results showed that the Transgelin-2 protein contained ten α helices and two β folds.The α helix is located at M12-N14,L21-K28,A35R50,R60-L67,G70-A79,M96-Y114,T118-D120,V125-G130,M133-R150,and R207-Q208,respectively.The β folds are located in I91-T95 and Q123-T124,respectively,and the rest are coil.6.SPR technique was used to prove that there was a binding relationship between Transgelin-2 protein and six small molecule compounds,including CAT-180,CAT179,CAT-044,CAT-042,CAT-040 and CAT-039,respectively.The equilibrium dissociation constants KD were:3.802×10-4 M,5.005×10-4 M,9.537×10-5 M,6.108×10-5 M,2.959×10-4 M and 8.084×10-5 M.7.ITC technique was used to demonstrate that Transgelin-2 protein was bound to the small molecule compound CAT-180 at 400 μm,and the equilibrium dissociation constant Kd value was(9.81±3.00)× 10-6 M.The other five small molecules were not detected to bind to Transgelin-2 protein at the concentration of 400 μm in ITC experiment.8.NMR titration was used to prove that the Transgelin-2 protein was bound to CAT180,and the chemical shift of amino acids of the transgelin-2 protein under the binding state was analyzed.The Chemical shift of some amino acid residues(G20,S22,E30,T47,R50,K89,Q106,A134,R150,S156,G157,F162,K165,L178,N183,1185,L187,Q188,G190,G202,1209 and L210)have been changed,and the main change region was concentrated in the C-terminal of transgelin-2 protein.The interfaces of T47,R50,S156 and G157 may be one of the specific binding interfaces between Transgelin-2 protein and CAT-180.Conclusion:1.The recombinant Transgelin-2 protein expressing stable isotopes of non-labeled,15N-labeled and 15N-13C was successfully induced,and the recombinant protein of stable isotopes of human Transgelin-2 protein was obtained for the first time,which provided a tool protein for subsequent mechanism study.2.Three-dimensional NMR was used to characterize the full-length solution structure of the Transgelin-2 protein.The chemical shift of 92%of the main chain was identified,and the secondary structure of the Transgelin-2 protein was calculated.3.SPR technology and ITC technology were used to study the binding relationship and strength between Transgelin-2 protein and small molecule compound of agonist.Both methods proved that Transgelin-2 was bound to small molecule compound CAT-180.4.NMR titration showed that there were 22 interaction sites between Transgelin-2 protein and CAT-180,and at least one specific binding interface was possible.