Biochemical and structural characterization of MUPP1-PDZ4 domain from Mus musculus

• Published in: Acta biochimica et biophysica Sinica Vol. 47; no. 3; pp. 199 - 206
• Main Authors: Zhu, Haili, Liu, Zexu, Huang, Yuxin, Zhang, Chao, Li, Gang, Liu, Wei
• Format: Journal Article
• Language: English
• Published: China 01-03-2015
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Crystallography, X-Ray; Mice; Models, Molecular; Molecular Sequence Data; Mus musculus; PDZ Domains; PDZ结构域; Protein Conformation; Protein Stability; Protein Structure, Secondary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid; Static Electricity; 小家鼠; 特异性蛋白质; 生化; 细胞骨架; 结构表征; 聚乙二醇400; 蛋白质相互作用; crystallization; MUPP1; PDZ domain
• ISSN: 1672-9145; 1745-7270
• DOI: 10.1093/abbs/gmv002

Specific protein-protein interactions are important for biological signal transduction. The postsy- naptic density-95, disc-large, and zonulin-1 （PDZ） domain is one of the most abundant protein inter- action 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 prolifer- ation. The study on PDZ domain of MUPP1 helps to understand the mechanisms and functions of MUPPI. 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 sub- cloned into a pET-vector and expressed in Escherichia coil 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） contain- ing 2% （v/v） polyethylene glycol 400, 0.1 M imidazole, and 24% （w/v） polyethylene glycol mono- ethyl ether 5000. Finally, the crystal was diffracted with 1.6A. resolution. The crystal structure showed that MUPP1-PDZ4 domain contained three m-helices and six 13-strands in the core. The GLGI motif, L562/A564 on the B-strand B, and H605N608/L612 on the a-helix B formed a PDZ bind- ing pocket which could bind to the C-terminal of the binding partners. This biochemical and struc- tural information will provide insights into how PDZ binds to its target peptide and the theoretical foundation for the function of MUPPI.