Gan, Z. Y. | Search bioRxiv

bioRxiv2023

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.

molecular biology↗

bioRxiv2020

Mechanism and inhibition of SARS-CoV-2 PLpro

Coronaviruses, including SARS-CoV-2, encode multifunctional proteases that are essential for viral replication and evasion of host innate immune mechanisms. The papain-like protease PLpro cleaves the viral polyprotein, and reverses inflammatory ubiquitin and anti-viral ubiquitin-like ISG15 protein modifications1,2. Drugs that target SARS-CoV-2 PLpro (hereafter, SARS2 PLpro) may hence be effective as treatments or prophylaxis for COVID-19, reducing viral load and reinstating innate immune responses3. We here characterise SARS2 PLpro in molecular and biochemical detail. SARS2 PLpro cleaves Lys48-linked polyubiquitin and ISG15 modifications with high activity. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. We further exploit two strategies to target PLpro. A repurposing approach, screening 3727 unique approved drugs and clinical compounds against SARS2 PLpro, identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors were able to inhibit SARS2 PLpro with high potency and excellent antiviral activity in SARS-CoV-2 infection models.

biochemistry↗