Study On The Target Selectivity And Structural Biology Of The Anti-inflammatory Peptide Hydrostatin-sn10 From Sea Snake Venom

BackgroundSea snakes are rich in anti-inflammatory molecules with potential medicinal value,whose functionary mechanism and action targets are still unclear.Hydrophis Cyanocinctus has been used as food and medicine from ancient times in China,since its venom is abundant with plenty of various bioactive proteins and polypeptides which have a good effect in relieving rheumatoid arthritis,joint pain and autoimmune inflammatory diseases.Because the physicochemical properties of the anti-inflammatory components in snake venom are close to each other,and the quantity of some constituents is very low,it is rather difficult to separate out single compounds by traditional chromatography for the study on their structure,function and action mechanism.Our research group previously via phage display library technology established the method of panning anti-inflammatory components targeting the human tumor necrosis factor receptor type I?TNFR1?from the venom gland of Hydrophis Cyanocinctus,and screened out an anti-inflammatory peptide Hydrostatin-SN1 of 22 amino acids.Subsequently we modified the peptide by sequence fragment truncation and obtained a 10-mer peptide Hydrostatin-SN10 with stronger specificity towards TNFR1.Hydrostatin-SN10 exhibited significant anti-inflammatory effect in animal models of collagen induced arthritis?CIA?and inflammatory bowel disease?IBD?and is expected to be developed into novel target-selective marine peptide drugs for the treatment of TNF receptors?TNFRs?-related inflammatory diseases.The interaction between TNF-?and its two receptors?TNFR1 and TNFR2?and the abnormal activation of downstream signaling pathways are important factors for the development of autoimmune diseases such as rheumatoid arthritis and ulcerative colitis.Hydrostatin-SN1 binds to both TNFR1 and TNFR2 and is able to antagonize the interaction of TNF-?with TNFR1/TNFR2,while Hydrostatin-SN10 had stronger affinity with TNFR1 and could inhibit TNFR1-mediated downstream NF-?B and MAPKs signaling pathways at a certain range of concentration in cell experiments.However,it has not yet been verified whether Hydrostatin-SN10 binds to TNFR2 and whether it can competitively inhibit the interaction of TNF-?with TNFRs.Moreover,the binding sites and binding modes of TNFR1 with Hydrostatin-SN1/SN10 remain unknown,and the polypeptide structure transformation as well as the alteration of amino acids in active sites from Hydrostatin-SN1 to Hydrostatin-SN10 are not clear.Therefore,to validate the receptor selectivity of Hydrostatin-SN10 and explore the binding complex conformation of Hydrostatin-SN10 with TNFR1,would lay an important foundation for elucidating the molecular mechanism of Hydrostatin-SN10 specifically antagonizing TNFR1 and exerting anti-inflammatory effects.ObjectiveTo confirm the TNFR1-selectivity of Hydrostatin-SN10,determine the three-dimensional structure of TNFR1-SN10 complex by the method of structural biology,and find the binding sites and key interacting amino acids between the peptide and receptor,which will help to clarify the molecular mechanism of selective antagonism of Hydrostatin-SN10 against TNFR1 and lay a biological basis for further structure modification of the peptide and drug development of new TNFR1-selective inhibitors.Methods1.Through multiple biomolecular interaction detecting techniques test the interaction of Hydrostatin-SN10 with TNFR1,TNFR2 and TNF-?,obtaining affinity constants,binding kinetics and thermal kinetic parameters to analyze the interaction patterns;observe whether Hydrostatin-SN10 can block or inhibit TNF-?binding with TNFR1/TNFR2 in competition assays.2.Construct the prokaryotic expression vector of the extracellular domain of human TNFR1?sTNFR1?in the way of Ligation-Independent Cloning?LIC?,express sTNFR1 in Escherichia coli and grope suitable conditions of protein inducible expression;purify the protein by affinity chromatography,ion exchange chromatography and size exclusion chromatography to produce large quantities of sTNFR1 protein with high purity.3.Use isothermal titration calorimetry?ITC?to identify the in vitro binding ability of recombinant sTNFR1 with TNF-?and investigate its biological activity.4.Utilize nano differential scanning fluorescence technique?nanoDSF?to search for the buffer condition in which target protein keeps the best thermal stability;incubate sTNFR1 with Hydrostatin-SN10 and perform large-scale initial crystallization conditions screening of sTNFR1-unliganded and sTNFR1-SN10 complexes using hanging drop method;optimize positive crystallization conditions and cultivate protein crystals of high quality.5.Express and purify ¹⁵N-¹³C-labeled sTNFR1 in M9 medium supplemented with isotope-labeled nitrogen source and carbon source;construct fusion expression vector of maltose binding protein?MBP?and Hydrostatin-SN10 to express ¹⁵N-¹³C-labeled peptide Hydrostatin-SN10.6.Via nuclear magnetic resonance?NMR?determine the two-dimensional ¹H-¹⁵N HSQC spectroscopy of ¹⁵N-sTNFR1 and ¹⁵N-SN10,then add the corresponding unlabeled ligand for two-dimensional titration,identify chemical shifts which reveal binding hot spots of sTNFR1 complexed with Hydrostatin-SN10;incubate ¹⁵N-¹³C-labeled protein and peptide and collect the NMR data to analyze the solution three-dimensional structure of TNFR1-SN10 complex.Results1.We validated the target specificity of Hydrostatin-SN10 by microscale thermophoresis?MST?,surface plasmon resonance?SPR?and isothermal titration calorimetry?ITC?.MST experiments showed that Hydrostatin-SN10 had specific binding with TNFR1 and the affinity K_D?equilibrium dissociation constant?value was 2.83?M;Hydrostatin-SN10 did not interact with TNFR2 or TNF-?.SPR results showed that Hydrostatin-SN10 displayed no specific binding trend with TNFR2 or TNF-?;Hydrostatin-SN10 could competitively inhibit the interaction between TNF-?and TNFR1,but had no effect on the binding of TNF-?to TNFR2.ITC data revealed that between Hydrostatin-SN10 and TNFR1 there existed interaction with the affinity of 2.29?M,while binding was not observed for TNFR2 and the peptide.2.The prokaryotic expression vectors pMCSG7-TNFR1 and pMCSG7-sumo-TNFR1of human TNFR1 extracellular domain were successfully constructed.The protein was expressed in E.coli BL21 cultured with LB medium,and the induction temperature,induction time and inducer concentration were optimized for large scale expression.A buffer system for denaturation and renaturation of target protein has been established,and the recombinant sTNFR1 protein was purified to above 97%successively through nickel affinity chromatography,cation exchange chromatography and gel filtration chromatography column.The protein yield was about 7.3 mg per liter of bacterial suspension.3.ITC test showed that the sTNFR1 protein expressed in vitro had a binding capacity of 154.8 nM with TNF-?,which was consistent with the reported literature and close to commercial protein.4.We applied nanoDSF to investigate the best buffer condition of target protein for crystallization:20 mM Tris-HCl pH7.5,150 mM NaCl,and the Tm value of sTNFR1 was77.67±0.05?in this buffer.The 96-well hanging drop plates were used for preliminary screening of crystallization conditions and we found numerous crystallization conditions of sTNFR1-unliganded and TNFR1-SN10 complex.A few conditions were optimized,and high quality crystals of sTNFR1-unliganded?0.2 mm×0.2 mm×0.1 mm?were harvested.At present,the crystal growth of TNFR1-SN10 complex is continuing to be optimized.5.We collected the X-ray diffraction data of sTNFR1 crystals and by molecular replacement method analyzed its high resolution?1.69??three-dimensional structure,which is in accordance with the previously reported crystal structure of TNFR1?PDB ID:1NCF?after superimposition.6.We used ¹⁵N-NH₄Cl and ¹³C-glucose in M9 medium and successfully expressed¹⁵N-¹³C-sTNFR1 protein.We constructed the MBP-peptide fusion expression vector pMCSG9-SN10,expressed and purified ¹⁵N-¹³C-labeled peptide SN10 used for NMR experiments.ConclusionOn the one hand,we have confirmed that the sea snake-derived peptide Hydrostatin-SN10 can specifically bind to TNFR1 but not interacted with TNFR2 or TNF-?,and selectively inhibit the interaction of TNF-?with TNFR1;on the other hand,the initial crystals of TNFR1 complexed with SN10 were obtained,which would lay a solid foundation for further structural biology exploration and bring insight into the anti-inflammatory mechanism of SN10.