Capturing the catalytic intermediates of parkin ubiquitination

• 成果类型: Article
• 署名作者: Connelly, Elizabeth M.; Rintala-Dempsey, Anne C.; Gundogdu, Mehmet; Freeman, E. Aisha; Koszela, Joanna; Aguirre, Jacob D.; Zhu, Grace; Kamaraine, Outi; Tadayon, Roya; Walden, Helen; Shaw, Gary S.
• 署名单位: Western University (University of Western Ontario); University of Glasgow
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-14409
• DOI: 10.1073/pnas.2403114121
• 发表日期: 2024-08-06
• 关键词: e3 ligase; pink1-dependent phosphorylation; structural basis; reveals; mechanism; mutations; insights; pink1; localization; RESTRICTION

摘要

Parkin is an E3 ubiquitin ligase implicated in early- onset forms of Parkinson's disease. It catalyzes a transthiolation reaction by accepting ubiquitin (Ub) from an E2 conjugating enzyme, forming a short- lived thioester intermediate, and transfers Ub to mitochondrial membrane substrates to signal mitophagy. A major impediment to the development of Parkinsonism therapeutics is the lack of structural and mechanistic detail for the essential, short- lived transthiolation intermediate. It is not known how Ub is recognized by the catalytic Rcat domain in parkin that enables Ub transfer from an E2 similar to Ub conjugate to the catalytic site and the structure of the transthiolation complex is undetermined. Here, we capture the catalytic intermediate for the Rcat domain of parkin in complex with ubiquitin (Rcat-Ub) and determine its structure using NMR- based chemical shift perturbation experiments. We show that a previously unidentified alpha- helical region near the Rcat domain is unmasked as a recognition motif for Ub and guides the C- terminus of Ub toward the parkin catalytic site. Further, we apply a combination of guided AlphaFold modeling, chemical cross- linking, and single turnover assays to establish and validate a model of full- length parkin in complex with UbcH7, its donor Ub, and phosphoubiquitin, trapped in the process of transthiolation. Identification of this catalytic intermediate and orientation of Ub with respect to the Rcat domain provides important structural insights into Ub transfer by this E3 ligase and explains how the previously enigmatic Parkinson's pathogenic mutation T415N alters parkin activity.