Oral Presentation 50th Lorne Proteins Conference 2025

Structure of human PINK1 stabilised at a novel mitochondrial translocase assembly (114817)

Sylvie Callegari 1 2 , Nicholas S Kirk 1 2 , Zhong Yan Gan 1 2 , Toby Dite 1 2 , Simon Cobbold 1 2 , Andrew Leis 1 2 , Laura Dagley 1 2 , Alisa Glukhova 1 2 3 4 , David Komander 1 2
  1. Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia
  2. Department of Medical Biology, University of Melbourne, Melbourne, Victoria, Australia
  3. Monash Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia
  4. Department of Biochemistry and Pharmacology, University of Melbourne, Melbourne, Victoria, Australia

PINK1 is a ubiquitin kinase that accumulates on the outer membrane of damaged mitochondria. Upon accumulation, PINK1 dimerises and trans-autophosphorylates, resulting in conformational changes that enable it to bind and phosphorylate ubiquitin. Together with the E3 ligase Parkin, the phospho-ubiquitin signal is amplified to trigger mitophagy. These processes critically depend on the initial stabilisation of PINK1 on the surface of damaged mitochondria, specifically at the site of the Translocase of the Outer Membrane (TOM complex). Previous studies investigating the activation mechanism of PINK1 have relied on the recombinantly expressed kinase domain of insect PINK1. Using single particle cryo-electron microscopy, we determine the structure of stabilised human PINK1 on the TOM complex to a resolution of 3.1 Å. This is the first structure of human PINK1 and resolves the passage of the PINK1 N-terminus through the TOM40 beta barrels. Surprisingly, the structure reveals an unusual assembly of two TOM complexes joined by a VDAC2 homodimer. This structure challenges previous models on the mode of ubiquitin phosphorylation by PINK1, as well as the notion of translocases as standalone units within the membrane. Furthermore, it not only provides an insight into how PINK1 is stabilised at the TOM complex, but also reveals long-sought-after insights into the passage of substrate proteins through the TOM complex during the fundamental process of mitochondrial import.