title: Rational design of stable and functional hirudin III mutants with lower antigenicity.
abstract: Hirudin is an inhibitor of thrombin and used as an effective anticoagulant, but has a potential to develop unacceptable immune responses. In this study, two computational tools were used to predict T-cell epitopes within Hirudin variant III (HVIII) sequence, and design mutations that would lessen its antigenicity. Homology models of native and mutant HVIII proteins (T4K, S9G, V21G, and V21K) were generated, and further used to assess their interactions with thrombin. The docking experiment showed that all mutants had a suitable pattern of interactions, with similar or lower interaction energies compared with the native protein. These complexes were subsequently subjected to molecular dynamics simulation. All mutants complexes had overall stable structures over simulation time, with RMSD, gyration radius, hydrogen bonds numbers, and accessible surface areas patterns that were comparable with the native HVIII over time. Interestingly, in all mutants, a shorter length was observed for the two salt bridges Arg73-Asp55 and Arg77-Glu57, which are suggested to be important in Hirudin-thrombin complex formation. Best selected mutants expressed in Escherichia coli BL21(DE3), subsequently SDS-PAGE and Western blot analysis confirmed the successful same expression of Hirudin and mutants. In conclusion, we believe that this computational approach could identify potentially safer proteins with preserved or even improved functionality.
authors: Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
authors: Saeme
authors: S
authors: Asgari
authors: Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran. Electronic address: mirzahoseini@pasteur.ac.ir.
authors: Hasan
authors: H
authors: Mirzahoseini
authors: Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
authors: Morteza
authors: M
authors: Karimipour
authors: Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
authors: Hamze
authors: H
authors: Rahimi
authors: Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran; Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran. Electronic address: aehabibi@sina.tums.ac.ir.
authors: Azadeh
authors: A
authors: Ebrahim-Habibi
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D015153:Blotting, Western; D019295:Computational Biology / Q000379:methods*; D015195:Drug Design; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli; D006684:HLA-DR Antigens / Q000276:immunology / Q000378:metabolism; D006629:Hirudins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D047709:Hirudo medicinalis / Q000235:genetics / Q000276:immunology*; D006801:Humans; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010314:Partial Thromboplastin Time; D017354:Point Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism
chemical_list: D000939:Epitopes; D006684:HLA-DR Antigens; D006629:Hirudins; D011993:Recombinant Fusion Proteins; D013917:Thrombin
keywords: Antigenicity; Deimmunization; Hirudin; Homology modeling; Molecular dynamics simulation; Structure–function analysis