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Journal of ZheJiang University(Medical Science)  2012, Vol. 41 Issue (2): 153-158    DOI: 10.3785/j.issn.1008-9292.2012.02.005
K83 site affects PICK1 PDZ binding ability
FENG Yong,QIAO Mu,LU Yu-ting,JIANG Ya-jian,WANG Na,ZHU Li-jun
Institute of neuroscience,Zhejiang University School of Medicine,Hangzhou 310058,China
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Abstract  Objective: To investigate the role of 83 site in interaction of GluR2 C-terminal and PICK1 PDZ domain.
Methods: Docking structure of PICK1 PDZ domain with GluR2 C terminal PDZ binding motif was built with computer software.After K83 site was substituted by other amino acid,the structure and binding energy were recalculated; meanwhile,site specific mutants were constructed using wild type full length cDNA as template.Mutants were co-transfected with GluR2 into HEK293T cells.After staining,the distribution of PICK1 and GluR2 were observed under confocal microscope.
Results: Wild type PICK1 and GluR2 formed many co-clusters in HEK293T cells as reported by other research groups; but different K83 mutant had different distribution in HEK293T cells.
Conclusions: The K83 site in PDZ domain of PICK1 is important for the interaction between PICK1 and GluR2.Altering lysine will probably change the hydrophobic interactions,the hydrogen bonds or the electrostatic interactions formed between PICK1 PDZ domain and GluR2 C terminal; accordingly,that will change the binding capacity between PICK1 and GluR2 in varying degrees.

Key wordsProtein kinase C      Protein conformation      Proteins/chem      Receptors,glutamate/metab      Computer simulation      PICK1      PDZ domain      GluR2 subunit      Mutagenesis     
Published: 25 March 2012
Cite this article:

. K83 site affects PICK1 PDZ binding ability. Journal of ZheJiang University(Medical Science), 2012, 41(2): 153-158.

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目的:研究PICK1(protein interacting with C kinase 1)蛋白PDZ结构域内83位点赖氨酸(K83) 对PICK1和 AMPA受体GluR2亚单位相互作用的影响。
方法:利用计算机对PICK1 PDZ结构域和GluR2 C末端4个氨基酸残基进行对接模拟,然后将K83进行虚拟点突变,计算并观察突变后结构和键能的改变。利用实验室已有的野生型全长PICK1 cDNA质粒为模板,构建点突变质粒,与野生型GluR2共转到HEK293T细胞,观察两者在细胞内定位和分布的改变。
结果:当野生型PICK1与GluR2共转染时,HEK293T细胞有大量PICK1 和GluR2共定位的集簇(cluster)。当我们把构建的PICK1突变体与GluR2共转染时,不同的突变体表现出不一样的改变。
结论:改变K83位点的氨基酸结构,很可能会改变PICK1 PDZ结构域与GluR2 C末端结合所形成的疏水、氢键、静电相互作用,使得PDZ结构域与GluR2 C末端的结合能力发生不同程度的改变。

关键词: 蛋白激酶C,  蛋白质构象,  蛋白质类/化学,  受体,  谷氨酸/代谢,  计算机模拟,  PICK1,  PDZ结构域,  GluR2亚基,  定点突变 
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