Wagner Julian P, Sauvé Véronique, Saran Anshu, Gehring Kalle
Department of Biochemistry, McGill University, Montreal, Quebec, Canada; Centre de Recherche en Biologie Structurale, McGill University, Montreal, Quebec, Canada.
Department of Biochemistry, McGill University, Montreal, Quebec, Canada; Centre de Recherche en Biologie Structurale, McGill University, Montreal, Quebec, Canada.
J Biol Chem. 2025 Jan;301(1):108051. doi: 10.1016/j.jbc.2024.108051. Epub 2024 Dec 3.
Mutations in the E3 ubiquitin ligase parkin cause a familial form of Parkinson's disease. Parkin and the mitochondrial kinase PTEN-induced kinase 1 assure quality control of mitochondria through selective autophagy of mitochondria (mitophagy). Whereas numerous parkin mutations have been functionally and structurally characterized, several Parkinson's disease mutations found in the catalytic Rcat domain of parkin remain poorly understood. Here, we characterize two pathogenic Rcat mutants, T415N and P437L. We demonstrate that both mutants exhibit impaired activity using autoubiquitination and ubiquitin vinyl sulfone assays. We determine the minimal ubiquitin-binding segment and show that both mutants display impaired binding of ubiquitin charged on the E2 enzyme. Finally, we use AlphaFold 3 to predict a model of the phospho-parkin:phospho-ubiquitin:ubiquitin-charged E2 complex. The model shows the repressor element of parkin and the N-terminal residues of the catalytic domain form a helix to position ubiquitin for transfer from the E2 to parkin. Our results rationalize the pathogenicity of the parkin mutations and deepen our understanding of the active parkin:E2∼Ub complex.
E3泛素连接酶帕金（Parkin）的突变会导致家族性帕金森病。帕金和线粒体激酶PTEN诱导激酶1通过线粒体的选择性自噬（线粒体自噬）确保线粒体的质量控制。尽管众多帕金突变已在功能和结构上得到表征，但在帕金的催化Rcat结构域中发现的一些帕金森病突变仍了解甚少。在此，我们对两个致病性Rcat突变体T415N和P437L进行了表征。我们通过自身泛素化和泛素乙烯砜测定证明这两个突变体均表现出活性受损。我们确定了最小的泛素结合片段，并表明这两个突变体对E2酶上负载的泛素的结合均受损。最后，我们使用AlphaFold 3预测磷酸化帕金：磷酸化泛素：负载泛素的E2复合物的模型。该模型显示帕金的阻遏元件和催化结构域的N端残基形成一个螺旋，以使泛素定位以便从E2转移至帕金。我们的结果解释了帕金突变的致病性，并加深了我们对活性帕金：E2∼Ub复合物的理解。
