Biochemical and structural characterization of MUPP1-PDZ4 domain from Mus musculus.
Biochemical and structural characterization of MUPP1-PDZ4 domain from Mus musculus.
复制标题
小家鼠 MUPP1-PDZ4 结构域的生化和结构特征。
DOI：
10.1093/abbs/gmv002
复制
发表时间：
2015-03
影响因子：
3.7
通讯作者：
Liu Wei
中科院分区：
文献类型：
--
作者：
Zhu Haili;Liu Zexu;Huang Yuxin;Zhang Chao;Li Gang;Liu Wei
特异性蛋白质-蛋白质相互作用对于生物信号转导是重要的。突触后密度-95、椎间盘-大和zonulin-1（PDZ）结构域是最丰富的蛋白质相互作用模块之一。多PDZ结构域蛋白1（Multi-PDZ-domainprotein 1，MUPP 1）作为一种支架蛋白，含有13个PDZ结构域，在细胞骨架结构、细胞极性和细胞增殖中起重要作用。对MUPP 1的PDZ结构域的研究有助于了解MUPP 1的作用机制和功能。本研究克隆、表达、纯化并鉴定了小家鼠MUPP 1的第四个PDZ结构域（MUPP 1-PDZ 4）。将MUPP 1-PDZ 4结构域亚克隆到pET载体中并在大肠杆菌中表达。采用亲和层析和分子排阻层析对蛋白进行纯化。MUPP 1-PDZ 4蛋白是溶液中摩尔质量为16.4 kDa的单体，熔点为60.3 ℃。使用坐滴气相扩散法，在含有2%（v/v）聚乙二醇400、0.1M咪唑和24%（w/v）聚乙二醇单乙醚5000的溶液（pH 7.0）中获得MUPP 1-PDZ 4蛋白质晶体。最后，以1.6 μ m的分辨率衍射晶体。晶体结构分析表明，MUPP 1-PDZ 4结构域的核心含有3个α螺旋和6个β链。GLGI基序、β链B上的L562/A564和α螺旋B上的H605/V608/L 612形成PDZ结合口袋，其可结合结合配偶体的C末端。这些生化和结构信息将为PDZ如何与其靶肽结合以及MUPP 1功能的理论基础提供见解。
Specific protein-protein interactions are important for biological signal transduction. The postsynaptic density-95, disc-large, and zonulin-1 (PDZ) domain is one of the most abundant protein interaction modules. Multi-PDZ-domain protein 1 (MUPP1), as a scaffold protein, contains 13 PDZ domains and plays an important role in cytoskeletal organization, cell polarity, and cell proliferation. The study on PDZ domain of MUPP1 helps to understand the mechanisms and functions of MUPP1. In the present study, the fourth PDZ domain of MUPP1 (MUPP1-PDZ4) from Mus musculus was cloned, expressed, purified, and characterized. The MUPP1-PDZ4 domain was subcloned into a pET-vector and expressed in Escherichia coli. Affinity chromatography and size-exclusion chromatography were used to purify the protein. MUPP1-PDZ4 protein was a monomer with a molar mass of 16.4 kDa in solution and had a melting point of 60.3°C. Using the sitting-drop vapor-diffusion method, MUPP1-PDZ4 protein crystals were obtained in a solution (pH 7.0) containing 2% (v/v) polyethylene glycol 400, 0.1 M imidazole, and 24% (w/v) polyethylene glycol monoethyl ether 5000. Finally, the crystal was diffracted with 1.6 Å resolution. The crystal structure showed that MUPP1-PDZ4 domain contained three α-helices and six β-strands in the core. The GLGI motif, L562/A564 on the β-strand B, and H605/V608/L612 on the α-helix B formed a PDZ binding pocket which could bind to the C-terminal of the binding partners. This biochemical and structural information will provide insights into how PDZ binds to its target peptide and the theoretical foundation for the function of MUPP1.
登录
查看更多内容
影响因子：
64.8
作者：
H. Lipson
通讯作者：
H. Lipson
影响因子：
4.4
作者：
Kundu K;Backofen R
通讯作者：
Backofen R
DOI：
10.1107/s2053230x13033712
发表时间：
2014
期刊：
Acta crystallographica. Section F, Structural biology communications
影响因子：
--
作者：
Hongpeng Wang;Yan Zhang;Zhenyi Zhang;W. Jin;Geng Wu
通讯作者：
Hongpeng Wang;Yan Zhang;Zhenyi Zhang;W. Jin;Geng Wu
影响因子：
7
作者：
Deng, MH;Mehta, S;Chen, T
通讯作者：
Chen, T
影响因子：
7
作者：
Minghua Deng;Shipra Mehta;Fengzhu Sun;Ting Chen
通讯作者：
Minghua Deng;Shipra Mehta;Fengzhu Sun;Ting Chen