Mutations in the E3 ubiquitin ligase parkin (PARK2, also known as PRKN) and the protein kinase PINK1 (also known as PARK6) are linked to autosomal-recessive juvenile parkinsonism (AR-JP)1,2; at the cellular level, these mutations cause defects in mitophagy, the process that organizes the destruction of damaged mitochondria3,4. Parkin is autoinhibited, and requires activation by PINK1, which phosphorylates Ser65 in ubiquitin and in the parkin ubiquitin-like (Ubl) domain. Parkin binds phospho-ubiquitin, which enables efficient parkin phosphorylation; however, the enzyme remains autoinhibited with an inaccessible active site5,6. It is unclear how phosphorylation of parkin activates the molecule. Here we follow the activation of full-length human parkin by hydrogen–deuterium exchange mass spectrometry, and reveal large-scale domain rearrangement in the activation process, during which the phospho-Ubl rebinds to the parkin core and releases the catalytic RING2 domain. A 1.8 Å crystal structure of phosphorylated human parkin reveals the binding site of the phospho-Ubl on the unique parkin domain (UPD), involving a phosphate-binding pocket lined by AR-JP mutations. Notably, a conserved linker region between Ubl and the UPD acts as an activating element (ACT) that contributes to RING2 release by mimicking RING2 interactions on the UPD, explaining further AR-JP mutations. Our data show how autoinhibition in parkin is resolved, and suggest a mechanism for how parkin ubiquitinates its substrates via an untethered RING2 domain. These findings open new avenues for the design of parkin activators for clinical use.Structural mass spectrometry of full-length human parkin and a structure of the activated parkin core reveal large-scale domain rearrangements involved in activation of parkin by PINK1.

中文翻译：

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PINK1激活parkin的机制

E3 泛素连接酶 parkin（PARK2，也称为 PRKN）和蛋白激酶 PINK1（也称为 PARK6）的突变与常染色体隐性遗传性幼年型帕金森症 (AR-JP)1,2 相关；在细胞水平上，这些突变会导致线粒体自噬缺陷，线粒体自噬是组织破坏受损线粒体的过程3、4。Parkin 是自动抑制的，需要 PINK1 激活，它使泛素和 parkin 泛素样 (Ubl) 结构域中的 Ser65 磷酸化。Parkin 结合磷酸泛素，从而实现有效的 parkin 磷酸化；然而，该酶仍然具有无法进入的活性位点 5、6 的自抑制状态。尚不清楚 parkin 的磷酸化如何激活该分子。在这里，我们通过氢-氘交换质谱法跟踪全长人类帕金的激活，并揭示了激活过程中的大规模结构域重排，在此过程中磷酸化 Ubl 重新结合到 parkin 核心并释放催化 RING2 结构域。磷酸化人类 parkin 的 1.8 Å 晶体结构揭示了磷酸化 Ubl 在独特 parkin 结构域 (UPD) 上的结合位点，涉及一个由 AR-JP 突变排列的磷酸盐结合袋。值得注意的是，Ubl 和 UPD 之间的保守连接区域充当激活元件 (ACT)，通过模拟 UPD 上的 RING2 相互作用来促进 RING2 的释放，从而解释了进一步的 AR-JP 突变。我们的数据显示了 parkin 中的自抑制是如何解决的，并提出了一种机制，即 parkin 如何通过不受限制的 RING2 域泛素化其底物。这些发现为设计用于临床的帕金激活剂开辟了新途径。