Identification of a Novel Zn2+-binding Domain in the Autosomal Recessive Juvenile Parkinson-related E3 Ligase Parkin
Identification of a Novel Zn2+-binding Domain in the Autosomal Recessive Juvenile Parkinson-related E3 Ligase Parkin
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DOI：
10.1074/jbc.m808700200
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发表时间：
2009-05-29
影响因子：
4.8
通讯作者：
Shaw, Gary S.
中科院分区：
文献类型：
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作者：
Hristova, Ventzislava A.;Beasley, Steven A.;Shaw, Gary S.
编码parkin蛋白的park2的错义突变约占常染色体隐性青少年帕金森病（ARJP）病例的50%。Parkin属于RBR (RING-between-RING) E3连接酶家族，参与泛素介导的Pael-R和synphillin-1等蛋白的降解和运输。基于序列相似性研究提出的parkin结构由n端泛素样结构域和c端RBR结构域组成。这些结构域由类似于160个残基的独特parkin序列分开，没有可识别的结构域结构。我们对细菌表达和纯化的parkin进行了有限的蛋白水解实验，在独特的parkin结构域序列中发现了一个新的结构域（RING0）。RING0在Cys(150)-Cys（169）和Cys(196)-His（215）之间由CX2-3CX11CX2C和CX4-6CX10-16-CX2（H/C）基序组成两个不同的保守的富含半胱氨酸的簇。该区域的半胱氨酸/组氨酸残基位置与parkin RING1和RING2结构域以及其他E3连接酶RING结构域相似。然而，在parkin中，一个26个残基的连接子区域将基序分开，这在其他RING结构中并不常见。此外，RING0结构域包括在parkin的泛素样区域和RBR区域之间的所有已知的ARJP突变位点。利用电喷雾电离质谱和电感耦合等离子体原子发射光谱分析，我们确定了RING0、RING1、IBR和RING2结构域分别结合2个Zn2+离子，这是首次观察到能够结合8个金属离子的E3连接酶。从parkin中去除锌会导致蛋白质几乎完全展开，这一观察结果合理地解释了在parkin中发现的基于半胱氨酸的ARJP突变，包括RING0 (C212Y)，它形成细胞包体和/或泛素化缺陷，可能是因为锌结合不良和错误折叠。parkin中RING0结构域的鉴定为该蛋白提供了一个新的整体结构域结构，这将对评估ARJP突变的作用和设计旨在了解该疾病的实验具有重要意义。
Missense mutations in park2, encoding the parkin protein, account for similar to 50% of autosomal recessive juvenile Parkinson disease (ARJP) cases. Parkin belongs to the family of RBR (RING-between-RING) E3 ligases involved in the ubiquitin-mediated degradation and trafficking of proteins such as Pael-R and synphillin-1. The proposed architecture of parkin, based largely on sequence similarity studies, consists of N-terminal ubiquitin-like and C-terminal RBR domains. These domains are separated by a similar to 160-residue unique parkin sequence having no recognizable domain structure. We used limited proteolysis experiments on bacterially expressed and purified parkin to identify a new domain (RING0) within the unique parkin domain sequence. RING0 comprises two distinct, conserved cysteine-rich clusters between Cys(150)-Cys(169) and Cys(196)-His(215) consisting of CX2-3CX11CX2C and CX4-6CX10-16-CX2(H/C) motifs. The positions of the cysteine/histidine residues in this region bear similarity to parkin RING1 and RING2 domains, as well as other E3 ligase RING domains. However, in parkin a 26-residue linker region separates the motifs, which is not typical of other RING domain structures. Further, the RING0 domain includes all but one of the known ARJP mutation sites between the ubiquitin-like and RBR regions of parkin. Using electrospray ionization mass spectrometry and inductively coupled plasma-atomic emission spectrometry analysis, we determined that the RING0, RING1, IBR, and RING2 domains each bind two Zn2+ ions, the first observation of an E3 ligase with the ability to bind eight metal ions. Removal of the zinc from parkin causes near complete unfolding of the protein, an observation that rationalizes cysteine-based ARJP mutations found throughout parkin, including RING0 (C212Y) that form cellular inclusions and/or are defective for ubiquitination likely because of poor zinc binding and misfolding. The identification of the RING0 domain in parkin provides a new overall domain structure for the protein that will be important in assessing the roles of ARJP mutations and designing experiments aimed at understanding the disease.