Interaction of PINK1 with nucleotides and kinetin

PINK1 is a ubiquitin kinase that accumulates on damaged mitochondria to trigger mitophagy, and PINK1 loss-of-function mutations cause early onset Parkinsons disease. Nucleotide analogues such as kinetin triphosphate (KTP) have been suggested to enhance PINK1 activity and may represent a therapeutic strategy for the treatment of Parkinsons disease. Here, we investigate the interaction of PINK1 with nucleotides, including KTP. We establish a cryo-EM platform exploiting the previously observed dodecamer assembly of Pediculus humanus corporis (Ph) PINK1 to determine PINK1 structures bound to AMP-PNP and ADP, which reveal unexpected conformational changes in the kinase N-lobe to 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. Mutation of the gatekeeper to Ala or Gly is required to enable PINK1 to bind and utilise KTP as a phosphate donor in ubiquitin phosphorylation and mitophagy. Indeed, HsPINK1 M318G can be used to conditionally uncouple PINK1 stabilisation and activity on mitochondria.