title: Structural basis of RGD-hirudin binding to thrombin: Tyr3 and five C-terminal residues are crucial for inhibiting thrombin activity.
abstract: BACKGROUND
  Hirudin is an anti-coagulation protein produced by the salivary glands of the medicinal leech Hirudomedicinalis. It is a powerful and specific thrombin inhibitor. The novel recombinant hirudin, RGD-hirudin, which contains an RGD motif, competitively inhibits the binding of fibrinogen to GPIIb/IIIa on platelets, thus inhibiting platelet aggregation while maintaining its anticoagulant activity.
  
  
  RESULTS
  Recombinant RGD-hirudin and six mutant variants (Y3A, S50A, Q53A, D55A, E57A and I59A), designed based on molecular simulations, were expressed in Pichia pastoris. The proteins were refolded and purified to homogeneity as monomers by gel filtration and anion exchange chromatography. The anti-thrombin activity of the six mutants and RGD-hirudin was tested. Further, we evaluated the binding of the mutant variants and RGD-hirudin to thrombin using BIAcore surface plasmon resonance analysis (SPR). Kinetics and affinity constants showed that the KD values of all six mutant proteins were higher than that of RGD-hirudin.
  
  
  CONCLUSIONS
  These findings contribute to a novel understanding of the interaction between RGD-hirudin and thrombin.
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, ministry of education, Fudan University, Shanghai, China. yinonghuang10@fudan.edu.cn.|The Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China. yinonghuang10@fudan.edu.cn.
authors.forename: Yinong
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, ministry of education, Fudan University, Shanghai, China. zhangyl@shmu.edu.cn.|The Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China. zhangyl@shmu.edu.cn.
authors.forename: Yanling
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, ministry of education, Fudan University, Shanghai, China. zhaobing828@163.com.|The Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China. zhaobing828@163.com.
authors.forename: Bing
authors.initials: B
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, ministry of education, Fudan University, Shanghai, China. qpxu10@fudan.edu.cn.|The Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China. qpxu10@fudan.edu.cn.
authors.forename: Qiping
authors.initials: Q
authors.lastname: Xu
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, ministry of education, Fudan University, Shanghai, China. mrhughwy@163.com.|The Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China. mrhughwy@163.com.
authors.forename: Xiushi
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, ministry of education, Fudan University, Shanghai, China. hysong@shmu.edu.cn.|The Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China. hysong@shmu.edu.cn.|Collaborative Innovation Center for Biotherapy, Sichuan University, Chengdu, China. hysong@shmu.edu.cn.
authors.forename: Houyan
authors.initials: H
authors.lastname: Song
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, ministry of education, Fudan University, Shanghai, China. minyu@shmu.edu.cn.|The Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China. minyu@shmu.edu.cn.
authors.forename: Min
authors.initials: M
authors.lastname: Yu
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, ministry of education, Fudan University, Shanghai, China. weimo1025@shmu.edu.cn.|The Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China. weimo1025@shmu.edu.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Mo
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D006629:Hirudins / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D058849:Protein Refolding; D020349:Surface Plasmon Resonance; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics*
chemical_list: D006629:Hirudins; C544959:RGD-hirudin; D014443:Tyrosine; D013917:Thrombin