Phosphorylation by PINK1 Releases the UBL Domain and Initializes the Conformational Opening of the E3 Ubiquitin Ligase Parkin

Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through targeted drug design.PINK1或PARKIN的功能丧失突变（Loss-of-function mutations）是常染色体隐性遗传性帕金森病（autosomal recessive Parkinson's disease）最常见的致病诱因。二者的基因编码产物——丝氨酸/苏氨酸激酶（Ser/Thr kinase）PINK1与E3泛素连接酶（E3 Ubiquitin ligase）Parkin，在线粒体质量控制通路（mitochondrial quality control pathway）中发挥协同功能。当线粒体遭受应激刺激时，PINK1可激活Parkin，促使其转位至受损线粒体并介导其泛素化（ubiquitination），进而推动受损线粒体从细胞中清除。尽管PINK1介导的Ser65位点磷酸化（phosphorylation of Ser65）是关键的起始步骤，但Parkin酶活性激活的分子机制（molecular mechanisms）仍有待阐明。本研究通过分子建模（molecular modeling）技术，构建了全原子分辨率（all atom resolution）的人类Parkin完整结构模型。在稳态（steady state）条件下，该泛素连接酶以闭合的自抑制构象（auto-inhibited conformation）维持失活状态，这一构象由分子内相互作用（intra-molecular interactions）所稳定。显然，Parkin若要释放其催化活性，必须经历大规模的结构重排（structural rearrangements）。为此，我们通过计算机模拟（in silico）构建了PINK1依赖的Ser65磷酸化模型，并首次开展了沿连续解折叠通路（sequential unfolding pathway）的Parkin构象分子动力学模拟（molecular dynamics simulation），该通路可解开其缠绕的结构域，使其催化活性得以释放。我们将无偏分子动力学模拟（free (unbiased) molecular dynamics simulation）、蒙特卡洛算法（Monte Carlo algorithms）与最小偏置方法（minimal-biasing methods），与基于细胞的高内涵成像（cell-based high content imaging）及生化实验（biochemical assays）相结合。研究发现，Ser65的磷酸化会使新定义的蛋白裂隙（cleft）加宽，并使具有调控作用的N端泛素样结构域（ubiquitin-like domain, UBL）发生解离。这一运动通过一系列进一步开放的构象传递，最终使负载泛素的E2辅酶（E2 co-enzyme）得以结合。随后两种酶催化中心（catalytic centers）的空间重定向，或可促进泛素基团（Ub moiety）的转移以激活Parkin。本项结构功能研究（structure-function study）为阐明具有神经保护作用（neuroprotective）的Parkin的调控机制与活性奠定了基础，该研究或可为通过靶向药物设计（targeted drug design）开发小分子Parkin激活剂（small molecule Parkin activators）开辟新的研究方向。