Structural Function Analysis Of Serralysin Protease MP And Its Inhibitor LupI

Protease is a class of enzymes that hydrolyzes proteins by cutting protein peptide bonds in vivo,controlling protein size,composition,spatial conformation,and its final degradation.The physiological activities and diseases in the organism are closely related to proteases,such as digestion and absorption of food,blood coagulation,hemolysis,anti-inflammatory,blood pressure regulation,cell differentiation,cancer metastasis,and activation of physiologically active peptides.Serralysin belongs to the subfamily of zinc metalloprotease M10B and secretes by a variety of pathogenic Gram-negative bacteria.It is a key virulence factor for some diseases.Serralysin inhibitor is effective drug for the treatment of these diseases and can prevent antibiotic resistance in pathogenic bacteria.The serralysin protease MP and its inhibitor LupI studied in this paper are all secreted by the marine microbial Flavobacterium sp.YS-80-122.We made the prokaryotic expression on the protease MP.Then purified the protease MP,inhibitor LupI and MP-LupI comlex,respectively.The crystals of free LupI and MP-LupI complex were subjected to crystallization condition screening and X-ray diffraction analysis.The results are as follows:Construction of prokaryotic expression vector of protease MP:The genomic DNA extraction kit was used to extract Flavobacterium sp.YS-80-122 genomic DNA as a template,and PCR amplification was carried out using a designed primer with a restriction site.First,the pTOPO-MP prokaryotic expression vector was constructed,and it was confirmed that MP can be transferred to the plasmid.The recombinant plasmid pET22b-MP was then constructed by using the expressed plasmids pET22b and pTOPO-MP,and transformed into E.coli DH5?amplification plasmid.The resulting recombinant plasmid was amplified by original primers.The expressed fragment was approximately 1443 bp,which was consistent with the expected MP gene size.Transformed the correct recombinant plasmid into E.coli BL21 competent cells and induced to express.The supernatant after resuspending the bacterial suspension was verified by SDS-PAGE gel electrophoresis and activity measurement.The results showed that the protein was about 48 kDa in size,the same as the predicted protein molecule mass.The activity assay also showed it had protease activity.Preparation and purification of MP-LupI complex:Purification of protease MP by affinity chromatography using a HisTrap HP column.The inhibitor LupI was purified by size exclusion chromatography and ion exchange chromatography.After electrophoresis,the two were mixed in an equimolar ratio to obtain the MP-LupI complex.Finally,purified MP-LupI complex by size exclusion chromatography to obtain an electrophoresis-purified,single-purification sample of about 95?L of MP-LupI complex with a protein content of 20 mg/mL.Screening of crystallization conditions of MP-LupI complex:The crystallization conditions of free LupI and MP-LupI complex were screened by hanging drop diffusion method on the 24-well plate.The crystallization kits were also used to perform crystallization.LupI was crystallized under conditions of 0.1 M NaCl,0.1 M BIS-TRIS pH 6.5,1.5 M?NH₄?₂SO₄.MP-LupI complex crystals were obtained under two conditions:0.1 M DL-Malic acid,pH 7.0,12%w/v PEG 3350 to obtain rhombohedral crystals of MP-LupI complex;0.2 M NaCl,0.1 M MES,pH 6.5,10%w/v PEG 4000 to obtain an prism crystal of the MP-LupI complex.X-ray diffraction results and refinement of the crystal:X-ray diffraction was carried out on the LupI crystals and the MP-LupI complex crystals selected in this experiment.LupI crystal obtained high quality diffraction data in SSRF,resolution up to 1.59?,its cell space is P 21 2121,cell parameters are A=34.04?,B=39.28?,c=62.57?,?=?=?=90°,The molecular replacement method was used to R_work 0.210 and R_free to0.218.MP-LupI complex crystal obtained high quality diffraction data with a resolution of 2?in the same way,the cell space is P 1 21 1,the cell parameters are A=51.66?,B=51.85?,C=102.14?,?=?=90°??=97.68°,finishing to R_work of 0.184,R_free of 0.230.Three-dimensional structure analysis of LupI and MP-LupI complex:From the three-dimensional structure diagram,the inhibitor LupI forms a compact seven-strand anti-parallel?-barrel with a simple top-bottom topology,inserted into the MP through the N-terminal long-chain structure.The zinc ion active center changed and achieved inhibition.Compared to homologous inhibitors,LupI lacks a?-sheet.But the?-helix extension replaces some structure effects,stabilizing the structure of the inhibitor.By comparing the MP-LupI complex with proteases and inhibitors,it is possible to clearly confirm that the site of the key change in the inhibition process is the zinc ion active center and the loop of the contact sites.The N-terminus of the inhibitor binds to the zinc ion center of the MP,deflecting the Tyr226 which controls the activity of the enzyme,and occupies the position where the protease binds to the substrate to achieve inhibition.The loop between the?-sheets3 and 4 of the inhibitor also forms a hydrogen bond with the MP,making the inhibitory effect more stable.In this thesis,the stereostructure of the MP-LupI complex was obtained by X-ray diffraction.The structure differences between the free protease and the free inhibitor were analyzed.The structure was also compared with the homologous inhibitor.It helps to deeply understand the interaction mechanism between serralysin protease and inhibitor,determine the key regions in the inhibition process and the region can be omitted,and provide reference for the design of specific protease inhibitor drugs in the future.