Evolutionary analysis of PINK1 reveals key roles of the N- and C-terminal extensions and TOM20 in folding its kinase domain
- 1McGill University
Abstract
PTEN-induced kinase 1 (PINK1) is a mitochondrial serine/threonine kinase that initiates ubiquitin-dependent mitophagy and is mutated in early-onset Parkinson's disease. Despite extensive characterization of insect PINK1 orthologues, obtaining soluble and catalytically active recombinant Homo sapiens PINK1 (HsPINK1) has remained challenging, limiting its biochemical and structural investigation. Here, we identify an evolutionary divergence in the mechanisms supporting PINK1 stability and activity. Comparative analysis of 48 metazoan PINK1 orthologues revealed that predicted binding to the mitochondrial import receptor TOM20 is predominantly a vertebrate feature and coincides with divergence of the PINK1 N- and C-terminal extensions (NTE and CTE). Consistent with this distinction, invertebrate PINK1 orthologues were purified as active kinases, whereas vertebrate orthologues lacked detectable ubiquitin kinase activity. NMR spectroscopy demonstrated that humanization of ten residues within the Tribolium castaneum (Tc)PINK1 NTE-CTE interface is sufficient to confer direct binding to human TOM20, while co-expression with TOM20 increased the activity of recombinant HsPINK1. Guided by these evolutionary differences, we engineered a chimeric PINK1 containing a predominantly human kinase domain supported by Tc-derived NTE-CTE elements. The resulting kinase was catalytically active and required compatible interactions between the NTE-CTE and kinase C-lobe for activity. The chimera enabled characterization of PINK1 variants and revealed R152W as a likely pathogenic variant. Together, our findings support a model in which the NTE and CTE act as intramolecular determinants of PINK1 kinase stability and suggest that vertebrate PINK1 has evolved an increased dependence on the mitochondrial import machinery to maintain a functional state.
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BibTeX
@misc{shomali2026evolutionary,
title = {{Evolutionary analysis of PINK1 reveals key roles of the N- and C-terminal extensions and TOM20 in folding its kinase domain}},
author = {T. Shomali and S. Veyron and J.-F. Trempe},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.05.749644},
url = {https://www.biorxiv.org/content/10.64898/2026.09.05.749644v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Evolutionary analysis of PINK1 reveals key roles of the N- and C-terminal extensions and TOM20 in folding its kinase domain
AU - T. Shomali
AU - S. Veyron
AU - J.-F. Trempe
PY - 2026
DA - 2026/09/05
PB - bioRxiv
DO - 10.64898/2026.09.05.749644
UR - https://www.biorxiv.org/content/10.64898/2026.09.05.749644v1
AB - PTEN-induced kinase 1 (PINK1) is a mitochondrial serine/threonine kinase that initiates ubiquitin-dependent mitophagy and is mutated in early-onset Parkinson's disease. Despite extensive characterization of insect PINK1 orthologues, obtaining soluble and catalytically active recombinant Homo sapiens PINK1 (HsPINK1) has remained challenging, limiting its biochemical and structural investigation. Here, we identify an evolutionary divergence in the mechanisms supporting PINK1 stability and activity. Comparative analysis of 48 metazoan PINK1 orthologues revealed that predicted binding to the mitochondrial import receptor TOM20 is predominantly a vertebrate feature and coincides with divergence of the PINK1 N- and C-terminal extensions (NTE and CTE). Consistent with this distinction, invertebrate PINK1 orthologues were purified as active kinases, whereas vertebrate orthologues lacked detectable ubiquitin kinase activity. NMR spectroscopy demonstrated that humanization of ten residues within the Tribolium castaneum (Tc)PINK1 NTE-CTE interface is sufficient to confer direct binding to human TOM20, while co-expression with TOM20 increased the activity of recombinant HsPINK1. Guided by these evolutionary differences, we engineered a chimeric PINK1 containing a predominantly human kinase domain supported by Tc-derived NTE-CTE elements. The resulting kinase was catalytically active and required compatible interactions between the NTE-CTE and kinase C-lobe for activity. The chimera enabled characterization of PINK1 variants and revealed R152W as a likely pathogenic variant. Together, our findings support a model in which the NTE and CTE act as intramolecular determinants of PINK1 kinase stability and suggest that vertebrate PINK1 has evolved an increased dependence on the mitochondrial import machinery to maintain a functional state.
ER -