Mutations in parkin and PINK1 cause early-onset Parkinson’s disease (EOPD). The ubiquitin ligase parkin is recruited to damaged mitochondria and activated by PINK1, a kinase that phosphorylates ubiquitin and the ubiquitin-like domain of parkin. Activated phospho-parkin then ubiquitinates mitochondrial proteins to target the damaged organelle for degradation. Here, we present the mechanism of activation of a new class of small molecule allosteric modulators that enhance parkin activity. The compounds act as molecular glues to enhance the ability of phospho-ubiquitin (pUb) to activate parkin. Ubiquitination assays and isothermal titration calorimetry with the most active compound (BIO-2007817) identify the mechanism of action. We present the crystal structure of a closely related compound (BIO-1975900) bound to a complex of parkin and two pUb molecules. The compound binds next to pUb on RING0 and contacts both proteins. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments confirm that activation occurs through release of the catalytic Rcat domain. In organello and mitophagy assays demonstrate that BIO-2007817 partially rescues the activity of parkin EOPD mutants, R42P and V56E, offering a basis for the design of activators as therapeutics for Parkinson’s disease.

Parkin 和 PINK1 突变会导致早发性帕金森病 (EOPD)。泛素连接酶 Parkin 被招募到受损的线粒体并被 PINK1 激活，PINK1 是一种磷酸化泛素和 Parkin 泛素样结构域的激酶。然后，激活的磷酸-parkin 泛素化线粒体蛋白，以靶向受损的细胞器进行降解。在这里，我们提出了增强 Parkin 活性的新型小分子变构调节剂的激活机制。这些化合物充当分子胶，增强磷酸泛素 (pUb) 激活 Parkin 的能力。使用最活跃的化合物 (BIO-2007817) 进行泛素化测定和等温滴定量热法确定了作用机制。我们展示了一种与 Parkin 和两个 pUb 分子复合物结合的密切相关化合物 (BIO-1975900) 的晶体结构。该化合物与 RING0 上的 pUb 结合并接触两种蛋白质。氢-氘交换质谱 (HDX-MS) 实验证实，活化是通过催化 Rcat 结构域的释放而发生的。细胞器和线粒体自噬测定表明，BIO-2007817 部分挽救了 Parkin EOPD 突变体 R42P 和 V56E 的活性，为设计帕金森病疗法的激活剂提供了基础。