Poster Presentation 49th Lorne Conference on Protein Structure and Function 2024

A structural understanding of PINK1 stabilisation and activation during mitochondrial damage (#409)

Sylvie Callegari 1 2 , Zhong Yan Gan 1 2 , Thanh Nguyen 1 3 , Nicholas Kirk 1 2 , Andrew Leis 1 2 , Grant Dewson 1 2 , Michael Lazarou 1 2 , 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. Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, 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. Loss of function mutations in PINK1 cause early onset Parkinson’s disease. We recently elucidated the ubiquitin kinase activation mechanism of Pediculus humanus corporis (Ph) PINK1 (1). A unique feature of PhPINK1 is that it can assemble into a stable dodecamer, well suited to cryo-EM analysis. Since the nucleotide analogue, kinetin triphosphate (KTP), has been suggested to enhance PINK1 activity and could represent a therapeutic strategy for the treatment of Parkinson’s disease, we exploited our PhPINK1 dodecamer as a platform to investigate the interaction of PINK1 with nucleotides. We have determined structures of PhPINK1 bound to AMP-PNP and ADP at resolutions of 2.84 Å and 3.13 Å respectively, revealing unexpected conformational changes in the kinase N-lobe that enable PINK1 to form a ubiquitin binding site. Strikingly, we find that KTP is unable to bind PhPINK1 or human (Hs) PINK1 due to a steric clash with the kinase ‘gatekeeper’ residue.

The use of PhPINK1 as a model has provided a deep insight into the mechanism of PINK1 ubiquitin kinase activity. However, 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). Using a crosslinking approach, we uncovered an unexpected interaction of PINK1 with components of the Translocase of the Inner Membrane 23 (TIM23) revealing that upon mitochondrial damage, the N-terminal region of PINK1 spans the import translocases on both the outer and inner mitochondrial membranes to generate a stable PINK1-TOM-TIM23 supercomplex. Structural studies are underway to elucidate the nature of this complex and determine the stabilisation mechanism of HsPINK1.

  1. Gan ZY, Callegari S, Cobbold SA, Cotton TR, Mlodzianoski MJ, Schubert AF, Geoghegan ND, Rogers KL, Leis A, Dewson G, Glukhova A, Komander D. Activation mechanism of PINK1. Nature. 2022 Feb;602:328-335.