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Autosomal recessive mutations in the PINK1 gene are causal for Parkinson's disease (PD). PINK1 encodes a mitochondrial localized protein kinase that is a master-regulator of mitochondrial quality control pathways. Structural studies to date have elaborated the mechanism of how mutations located within the kinase domain disrupt PINK1 function; however, the molecular mechanism of PINK1 mutations located upstream and downstream of the kinase domain is unknown. We have employed mutagenesis studies to define the minimal region of human PINK1 required for optimal ubiquitin phosphorylation, beginning at residue Ile111. Inspection of the AlphaFold human PINK1 structure model predicts a conserved N-terminal α-helical extension (NTE) domain forming an intramolecular interaction with the C-terminal extension (CTE), which we corroborate using hydrogen/deuterium exchange mass spectrometry of recombinant insect PINK1 protein. Cell-based analysis of human PINK1 reveals that PD-associated mutations (e.g. Q126P), located within the NTE : CTE interface, markedly inhibit stabilization of PINK1; autophosphorylation at Serine228 (Ser228) and Ubiquitin Serine65 (Ser65) phosphorylation. Furthermore, we provide evidence that NTE and CTE domain mutants disrupt PINK1 stabilization at the mitochondrial Translocase of outer membrane complex. The clinical relevance of our findings is supported by the demonstration of defective stabilization and activation of endogenous PINK1 in human fibroblasts of a patient with early-onset PD due to homozygous PINK1 Q126P mutations. Overall, we define a functional role of the NTE:CTE interface towards PINK1 stabilization and activation and show that loss of NTE : CTE interactions is a major mechanism of PINK1-associated mutations linked to PD.

Human autosomal recessive mutations in the PINK1 gene are causal for Parkinson’s disease (PD). PINK1 encodes a mitochondrial localised protein kinase that is a master-regulator of mitochondrial quality control pathways. Structural studies to date have elaborated the mechanism of how mutations located within the kinase domain disrupt PINK1 function, however, the molecular mechanism of PINK1 mutations located upstream and downstream of the kinase domain are unknown. We have employed mutagenesis studies of human PINK1 in cells to define the minimal region of PINK1, required for optimal ubiquitin phosphorylation, beginning at residue Ile111. Bioinformatic analysis of the region spanning Ile111 to the kinase domain and inspection of the AlphaFold human PINK1 structure model predicts a conserved N-terminal a-helical domain extension (NTE domain) within this region corroborated by hydrogen/deuterium exchange mass spectrometry (HDX-MS) of recombinant insect PINK1 protein. The AlphaFold structure also predicts the NTE domain forms an intramolecular interaction with the C-terminal extension (CTE). Cell-based analysis of human PINK1 reveals that PD-associated mutations (e.g. Q126P), located within the NTE:CTE interface, markedly inhibit stabilization of PINK1; autophosphorylation at Serine228 (Ser228); and Ubiquitin Serine65 (Ser65) phosphorylation. Furthermore, we provide evidence that NTE domain mutants do not affect intrinsic catalytic kinase activity but do disrupt PINK1 stabilisation at the mitochondrial Translocase of outer membrane (TOM) complex. The clinical relevance of our findings is supported by the demonstration of defective stabilization and activation of endogenous PINK1 in human fibroblasts of a patient with early-onset PD due to homozygous PINK1 Q126P mutations. Overall, we define a functional role of the NTE:CTE interface towards PINK1 stabilisation and activation and show that loss of NTE:CTE interactions is a major mechanism of PINK1-associated mutations linked to PD. Description of files Figure 1B PINK1 First 16 lanes. Other lanes are from unrelated experiments Lane 1: Marker, Lane 2-3: hPINK1 WT (-CCCP), Lane 4-5: hPINK1 WT (+CCCP 3 hour), Lane 6-7: hPINK1 KI (-CCCP), Lane 8-9: hPINK1 KI (+CCCP 3 hour), Lane 10-11: OPA3111hPINK1 (-CCCP), Lane 12-13: OPA3111hPINK1 (+CCCP 3 hour), Lane 14-15: HSP60111hPINK1 (-CCCP), Lane 16-17: HSP60111hPINK1 (+CCCP 3 hour) OPA1 (High exposure) Lane 1: Marker, Lane 2-3: hPINK1 WT (-CCCP), Lane 4-5: hPINK1 WT (+CCCP 3 hour), Lane 6-7: hPINK1 KI (-CCCP), Lane 8-9: hPINK1 KI (+CCCP 3 hour), Lane 10-11: OPA3111hPINK1 (-CCCP), Lane 12-13: OPA3111hPINK1 (+CCCP 3 hour), Lane 14-15: HSP60111hPINK1 (-CCCP), Lane 16-17: HSP60111hPINK1 (+CCCP 3 hour) GAPDH (Low exposure) Lane 1: Marker, Lane 2-3: hPINK1 WT (-CCCP), Lane 4-5: hPINK1 WT (+CCCP 3 hour), Lane 6-7: hPINK1 KI (-CCCP), Lane 8-9: hPINK1 KI (+CCCP 3 hour), Lane 10-11: OPA3111hPINK1 (-CCCP), Lane 12-13: OPA3111hPINK1 (+CCCP 3 hour), Lane 14-15: HSP60111hPINK1 (-CCCP), Lane 16-17: HSP60111hPINK1 (+CCCP 3 hour) Figure 1C Figure 1C OPA1: Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 3 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 3 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 3 hour), Lane 14-15: OPA111hPINK1 (-CCCP), Lane 16-17: OPA111hPINK1 (+CCCP 3 hour), Lane 18-19: HSP60111hPINK1 (-CCCP), Lane 20-21: HSP60111hPINK1 (+CCCP 3 hour) Figure 1C Parkin: Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 3 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 3 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 3 hour), Lane 14-15: OPA111hPINK1 (-CCCP), Lane 16-17: OPA111hPINK1 (+CCCP 3 hour), Lane 18-19: HSP60111hPINK1 (-CCCP), Lane 20-21: HSP60111hPINK1 (+CCCP 3 hour) Figure 1C PINK1: Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 3 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 3 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 3 hour), Lane 14-15: OPA111hPINK1 (-CCCP), Lane 16-17: OPA111hPINK1 (+CCCP 3 hour), Lane 18-19: HSP60111hPINK1 (-CCCP), Lane 20-21: HSP60111hPINK1 (+CCCP 3 hour) Figure 1C pS65 Parkin: Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 3 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 3 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 3 hour), Lane 14-15: OPA111hPINK1 (-CCCP), Lane 16-17: OPA111hPINK1 (+CCCP 3 hour), Lane 18-19: HSP60111hPINK1 (-CCCP), Lane 20-21: HSP60111hPINK1 (+CCCP 3 hour) Figure 1C pUb: Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 3 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 3 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 3 hour), Lane 14-15: OPA111hPINK1 (-CCCP), Lane 16-17: OPA111hPINK1 (+CCCP 3 hour), Lane 18-19: HSP60111hPINK1 (-CCCP), Lane 20-21: HSP60111hPINK1 (+CCCP 3 hour) Figure 1C Vinculin: Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 3 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 3 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 3 hour), Lane 14-15: OPA111hPINK1 (-CCCP), Lane 16-17: OPA111hPINK1 (+CCCP 3 hour), Lane 18-19: HSP60111hPINK1 (-CCCP), Lane 20-21: HSP60111hPINK1 (+CCCP 3 hour) Figure 1D Figure 1D OPA1, PINK1 and GAPDH: Upper left: blot 1: Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O) (not shown in paper), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) (not shown in paper), Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour). Lower left blot 2: Lane 1: Marker, Lane 2-3: 111PINK1(111-581) (-A/O), Lane 4-5: 111PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: PINK1(1-34)-PINK1(111- 581), (-A/O), Lane 16-17: PINK1(1-34)-PINK1(111- 581), (+A/O 9 hour). Upper right blot 3: Lane 1: Marker, Lane 2-3: pOTC(1-30)-PINK1(111-581) (-A/O), Lane 4-5: pOTC(1-30)-PINK1(111-581) (+A/O 9 hour), Lane 6-7: pOTC(1-30)-(L5A/L8A/L9A) PINK1(111-581) (-A/O), Lane 8-9: pOTC(1-30)-(L5A/L8A/L9A) PINK1(111-581) (+A/O 9 hour), Lane 10: pOTC(1-30)-(L5A/L8A/L9A) PINK1(111-581) (+A/O 9 hour), (not shown in paper), Lane 11-12: pF1ATPase(1-30)- PINK1(111-581) (-A/O), Lane 13-14: pF1ATPase(1-30)- PINK1(111-581) (+A/O 9 hour) lane 15-16: pF1- b ATPase (W29A/C32A/M33A) (1-30)- PINK1(111-581) (-A/O), lane 17-18: pF1- b ATPase (W29A/C32A/M33A) (1-30)- PINK1(111-581) (+A/O 9 hour). Lower right: blot 4: (not shown in paper) Lane 1: Marker, Lane 2-3: pALDH(1-19)-PINK1(111-581) (-A/O), Lane 4-5: pALDH(1-19)-PINK1(111-581) (+A/O 9 hour), Lane 6-7: pALDH (1-19) -(L15A) PINK1(111-581) (-A/O), Lane 8-9: pALDH (1-19) -(L15A) PINK1(111-581) (+A/O 9 hour. Figure 1D pUb: Upper left: blot 1: Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O) (not shown in paper), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) (not shown in paper), Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour). Lower left blot 2: Lane 1: Marker, Lane 2-3: 111PINK1(111-581) (-A/O), Lane 4-5: 111PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: PINK1(1-34)-PINK1(111- 581), (-A/O), Lane 16-17: PINK1(1-34)-PINK1(111- 581), (+A/O 9 hour). Upper right blot 3: Lane 1: Marker, Lane 2-3: pOTC(1-30)-PINK1(111-581) (-A/O), Lane 4-5: pOTC(1-30)-PINK1(111-581) (+A/O 9 hour), Lane 6-7: pOTC(1-30)-(L5A/L8A/L9A) PINK1(111-581) (-A/O), Lane 8-9: pOTC(1-30)-(L5A/L8A/L9A) PINK1(111-581) (+A/O 9 hour), Lane 10: pOTC(1-30)-(L5A/L8A/L9A) PINK1(111-581) (+A/O 9 hour), (not shown in paper), Lane 11-12: pF1ATPase(1-30)- PINK1(111-581) (-A/O), Lane 13-14: pF1ATPase(1-30)- PINK1(111-581) (+A/O 9 hour) lane 15-16: pF1- b ATPase (W29A/C32A/M33A) (1-30)- PINK1(111-581) (-A/O), lane 17-18: pF1- b ATPase (W29A/C32A/M33A) (1-30)- PINK1(111-581) (+A/O 9 hour). Lower right: blot 4: (not shown in paper) Lane 1: Marker, Lane 2-3: pALDH(1-19)-PINK1(111-581) (-A/O), Lane 4-5: pALDH(1-19)-PINK1(111-581) (+A/O 9 hour), Lane 6-7: pALDH (1-19) -(L15A) PINK1(111-581) (-A/O), Lane 8-9: pALDH(1-19)-(L15A) PINK1(111-581) (+A/O 9 hour). Figure 2B Figure 2B pUb: Upper left: blot 1: Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour). Upper right blot 2: Lane 1: Marker, Lane 2-3: 111PINK1(111-581) (-A/O), Lane 4-5: 111PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(115- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(115- 581), (+A/O 9 hour). Lower left blot 3: Lane 1: Marker, Lane 2-3: HSP60 (1-27)-PINK1(120- 581) (-A/O), Lane 4-5: HSP60 (1-27)-PINK1(120- 581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(125- 581) (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(125- 581) (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(130- 581) (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(130- 581) (+A/O 9 hour). Figure 2B PINK1: Upper left: blot 1: Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour). Upper right blot 2: Lane 1: Marker, Lane 2-3: 111PINK1(111-581) (-A/O), Lane 4-5: 111PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(115- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(115- 581), (+A/O 9 hour). Lower left blot 3: Lane 1: Marker, Lane 2-3: HSP60 (1-27)-PINK1(120- 581) (-A/O), Lane 4-5: HSP60 (1-27)-PINK1(120- 581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(125- 581) (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(125- 581) (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(130- 581) (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(130- 581) (+A/O 9 hour). Figure 2B OPA1: Upper left: blot 1: Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour). Upper right blot 2: Lane 1: Marker, Lane 2-3: 111PINK1(111-581) (-A/O), Lane 4-5: 111PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(115- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(115- 581), (+A/O 9 hour). Lower left blot 3: Lane 1: Marker, Lane 2-3: HSP60 (1-27)-PINK1(120- 581) (-A/O), Lane 4-5: HSP60 (1-27)-PINK1(120- 581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(125- 581) (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(125- 581) (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(130- 581) (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(130- 581) (+A/O 9 hour). Figure 2B GAPDH: Upper left: blot 1: Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour). Upper right blot 2: Lane 1: Marker, Lane 2-3: 111PINK1(111-581) (-A/O), Lane 4-5: 111PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(115- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(115- 581), (+A/O 9 hour). Lower left blot 3: Lane 1: Marker, Lane 2-3: HSP60 (1-27)-PINK1(120- 581) (-A/O), Lane 4-5: HSP60 (1-27)-PINK1(120- 581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(125- 581) (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(125- 581) (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(130- 581) (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(130- 581) (+A/O 9 hour). Figure 2C Figure 2C pUb: Blot 1: Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour). Blot 2: Lane 1: Marker, Lane 2-3: 111PINK1(111-581) (-A/O), Lane 4-5: 111PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(112- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(112- 581), (+A/O 9 hour). Blot 3: Lane 1: Marker, Lane 2-3: HSP60 (1-27)-PINK1(113- 581) (-A/O), Lane 4-5: HSP60 (1-27)-PINK1(113- 581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(114- 581) (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(114- 581) (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(115-581) (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(115- 581) (+A/O 9 hour). Figure 2C PINK1: Blot 1: Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour). Blot 2: Lane 1: Marker, Lane 2-3: 111PINK1(111-581) (-A/O), Lane 4-5: 111PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(112- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(112- 581), (+A/O 9 hour). Blot 3: Lane 1: Marker, Lane 2-3: HSP60 (1-27)-PINK1(113- 581) (-A/O), Lane 4-5: HSP60 (1-27)-PINK1(113- 581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(114- 581) (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(114- 581) (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(115-581) (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(115- 581) (+A/O 9 hour). Figure 2C OPA1: Blot 1: Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour). Blot 2: Lane 1: Marker, Lane 2-3: 111PINK1(111-581) (-A/O), Lane 4-5: 111PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(112- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(112- 581), (+A/O 9 hour). Blot 3

Autosomal recessive mutations in the PINK1 gene are causal for Parkinson's disease (PD). PINK1 encodes a mitochondrial localized protein kinase that is a master-regulator of mitochondrial quality control pathways. Structural studies to date have elaborated the mechanism of how mutations located within the kinase domain disrupt PINK1 function; however, the molecular mechanism of PINK1 mutations located upstream and downstream of the kinase domain is unknown. We have employed mutagenesis studies to define the minimal region of human PINK1 required for optimal ubiquitin phosphorylation, beginning at residue Ile111. Inspection of the AlphaFold human PINK1 structure model predicts a conserved N-terminal ��-helical extension (NTE) domain forming an intramolecular interaction with the C-terminal extension (CTE), which we corroborate using hydrogen/deuterium exchange mass spectrometry of recombinant insect PINK1 protein. Cell-based analysis of human PINK1 reveals that PD-associated mutations (e.g. Q126P), located within the NTE : CTE interface, markedly inhibit stabilization of PINK1; autophosphorylation at Serine228 (Ser228) and Ubiquitin Serine65 (Ser65) phosphorylation. Furthermore, we provide evidence that NTE and CTE domain mutants disrupt PINK1 stabilization at the mitochondrial Translocase of outer membrane complex. The clinical relevance of our findings is supported by the demonstration of defective stabilization and activation of endogenous PINK1 in human fibroblasts of a patient with early-onset PD due to homozygous PINK1 Q126P mutations. Overall, we define a functional role of the NTE:CTE interface towards PINK1 stabilization and activation and show that loss of NTE : CTE interactions is a major mechanism of PINK1-associated mutations linked to PD.