大鼠MUPP1蛋白PDZ8结构域的生化及晶体结构特性（英文）

Crystal Structure and Biochemical Characteristics of MUPP1 PDZ8 Domain from Rattus norvegicus

多重PDZ结构域蛋白1型(MUPP1)是一种存在于上皮细胞和神经细胞内含有13个PDZ结构域的重要支架蛋白.在上皮细胞中,MUPP1蛋白在紧密连接结构的形成和上皮细胞的极化过程中发挥重要作用.而在中枢神经系统中,MUPP1基因的1个提前终止突变导致了其最后12个PDZ结构域的缺失,以及严重的先天性脑积水.此外,MUPP1蛋白的表达水平与酒精依赖性和药物戒断的严重性也具有显著的相关性.因此,对MUPP1蛋白所含的PDZ结构域进行纯化和性质鉴定,将有助于深入研究MUPP1蛋白的功能和分子机制.在本文研究中,利用亲和纯化和分子筛技术,对大鼠来源的MUPP1蛋白的第8个PDZ结构域进行了表达和纯化.多角度激光光散射的数据表明:MUPP1-PDZ8结构域在溶液中为单体,分子量为16.4 k D.圆二色谱结果表明,MUPP1-PDZ8结构域具有较好的二级结构折叠,测得其熔解温度为71.6摄氏度,暗示该PDZ结构域在溶液中非常稳定.最后,MUPP1-PDZ8结构域的晶体结构显示,该结构域属于I型PDZ结构域,包含3个α螺旋和6个β折叠.其中GLGL模块、β折叠B上的1 351位亮氨酸,以及α螺旋B上的1 405位异亮氨酸/1 398位组氨酸形成的PDZ结合口袋,可以特异性地与其目标蛋白质的羧基末端相结合.综上所述,本文的研究提供了MUPP1-PDZ8结构域的生化特性,以及该结构域与其目标蛋白质相互作用的分子机制,这将为MUPP1蛋白的功能研究提供生物化学与结构生物学的理论基础.

Multi-PDZ domain protein 1（ MUPP1） is an important scaffold protein in epithelial cells and neuronal cells with 13 PDZ domains. In epithelial cells,MUPP1 is well known to be responsible for the organization of tight junctions（ TJs） and epithelial cell polarity. In the central nervous system,a truncating mutation in MUPP1 gene lacking of the last 12 PDZ domains causes severe congenital hydrocephalus. In addition, the MUPP1 expression level significantly associated with the alcohol dependence and the severities to drug withdrawal. Hence,to purify and characterize the PDZ domains of MUPP1 can be helpful for better understanding the molecular mechanism and function of MUPP1. In this study,the eighth PDZ domain of MUPP1 from Rattus norvegicus was expressed and purified by the affinity purification and size exclusion chromatography. The multi-angle laser light scattering data showed that the MUPP1-PDZ8 of 13. 4 k D was a monomer in solution. CD（ circular dichroism） spectrometry showed that the MUPP1-PDZ8 was well folded with a melting temperature of 71. 6 ℃,indicating it was stable in soluble forms. Finally,the crystal structure revealed that the MUPP1-PDZ8 belonged to type-I PDZ domain with three α-helices and six β-strands. The GLGL motif,L1351 on the β-strand B and the I1405/H1398 on theα-helix B formed the PDZ binding pocket to bind the C-terminus of the targets. Taken together,our studies provided the biochemical characteristics and binding mechanisms of MUPP1-PDZ8 domain,therefore offering the biochemical and structural basis for the functional studies of MUPP1 protein.