Epitopes Prediction And Inhibition Mechanism Ananlysis Of Proteins By Homology Modeling

The three-dimensional structural information of proteins is important for the study of function and mechanism of proteins. However, some protein crystallization is difficult to obtain, homology modeling is an important method to solve this problem. The crystal structure of protein, which sequence has identity over than30%with target protein and its crystal data has been published, was used as template for homology modeling.3D structure of target protein was constructed using homology modeling, and then active pocket, specific amino acids and inhibition mechanism were analyzed. This article focused on three proteins which were derived different sources and also difficult to crystallization. Their3D structures were separately obtained by homology modeling and specific3D structure information was separately analyzed of each protein.1. Homology modeling and B cell antigenic epitopes predicition of defibrase from Gloydious ussuriensis snake venomThe defibrase is the national essential drugs for the treatment of thromboembolic disease. However, this protein was obtained from snake venom, and it is a heterologous protein. Moreover, it is a macromolecule with over30KDa of molecular weight. Therefore, this protein drug is immunogenic and can cause allergic reactions. In this work, B cell linear epitopes were predicted using DNAStar soft ware and ABCpred、BCPREDS and other web servers. B-cell linear epitopes in defibrase were predicted to locate in the regions of27～35发、44～50、66～72、79～98、101～109、117～127、134～142、151～164、179～188、194～206、221～233and247～260. Moreover, using protein C activator from Agkistrodon contortrix contortrix venom (ACC-C) as template, a3D structure model of defibrase was constructed by Pythonwin program using modeling package MODELLER9.10. Stereochemical quality of the selected model was evaluated using PROCHECK, Errat and PROSA programs. The conformational epitopes of defibrase was predicted by3D structure of homology modeling combined with DiscoTope1.2server and cons PPISP online tools. B-cell conformational epitopes in defibrase were predicted to locate in the regions of45～51、81～90、95～106、134～14、153～159、177～182、225～232and251～257.2. Homology modeling and inhibitory mechanisms analysis of pectin methylesterase from Aspergillus flavusThe pectin methylesterase from Aspergillus flavus (AFPME) is the key enzyme secreted by Aspergillus flavus during plant invasion, and this enzyme is thought to be a virulence factor of phytopathogens. Plants do not product the active substances to inhibit the activity of this enzyme. Therefore, the application of exogenous inhibitors of AFPME may offer an alternative way to control the invasion of Aspergillus flavus.Previous studies of my lab found that epigallocatechin gallate (EGCG) of green tea strongly inhibited the activity of recombinant AFPME, however,(-)-epigallocatechin (EGC) of green tea was not obviously inhibit the activity of recombinant AFPME. In order to better understand the inhibition mechanism of this enzyme, a3D structure model of AFPME was constructed by using tomato fruit (Lycopersicon esculentum) as template. From the docking results, it was clear that EGCG could stably exist in the active pocket by hydrogen bonds and π-π interactions with Gln122and Trp228, respectively, that these two residues are located in the active site of enzyme. EGC has caused weak interactions through hydrogen bonds with some amino acid residues which are not located in the active site of enzyme, and EGC was not able to cover the active pocket of enzyme.3. Recombinant production, homology modeling and inhibition mechanism analysis of human chemokine (C-C motif) ligand1(CCL-1)The human chemokine (C-C motif) ligand1(CCL-1) is a key factor of inflammation, cancer, neuropathic pain and other diseases. The compounds which inhibited the activity of CCL1could be a drug candidate for treatment of these diseases. Here, the human CCL1was expressed in E.coli BL21(DE3), and it was found that CCL1was formed as inclusion body. After denaturing by6M urea and one-step purification by affinity chromatography, the purified CCL1was appeared as a single band in SDS-PAGE. The protein was then refolding in a buffer containing reduced/oxidized glutathione. The3D model of CCL1was built by homology modeling using CCL14crystal structure as template. The small molecular compound No. XL-10had good inhibiting effect by molecular docking and in vitro activity tests. The results of docking indicated that the inhibitor XL-10formed hydrogen bond with Asn30. Thr-31and Ser-32of CCL1.