Extended Data Fig. 5: Structural and biochemical analysis of the PhPINK1 dimer.

a, Comparison between PhPINK1 autophosphorylation and ubiquitin phosphorylation resolved previously (PDB: 6EQI)¹⁰, with relevant details in the active site (top row) and in the activation segment (bottom row). The right panel shows substrate disposition in relation to a modelled ATP molecule as in Fig. 3b. The dimeric PhPINK1 autophosphorylation complex appears to place Ser202 in an ideal phospho-accepting position. b, Comparison of activation segment structures in all published PINK1 structures, revealing high structural similarity. Ser375 (PhPINK1)/Ser377 (TcPINK1) is shown in ball-and-stick representation; this residue was mutated to Asp in one prior structure (see Extended Data Fig. 1a). c, PhPINK1 Ser375 corresponds to Ser402 in HsPINK1, which is a reported phosphorylation site^31,33, but is located within the activation segment and out of reach of the substrate-binding site within dimeric PhPINK1. Our structure does not reveal how autophosphorylation at this residue could be facilitated in cis or trans (left), nor how phosphorylation through e.g. an upstream kinase would contribute to PINK1 activity or function, since phosphorylation would likely disrupt the dimer (right). ATP was modelled as in Fig. 3b. d, Manual docking of dimeric PhPINK1 onto a cryo-EM structure of dimeric human TOM complex (PDB: 7CK6)⁶¹. The PhPINK1 dimer was oriented with its two N-helices (spanning ~80 Å) aligning with the two TOM7 subunits of the TOM complex dimer. TOM7 has been reported as essential for PINK1 stabilization on the TOM complex^27,28. Note that some TOM components have considerable cytosolic domains that would need to be accommodated in addition to a PINK1 dimer. e, Unidentified density in the 2.35 Å cryo-EM map connects Cys169 in the dimer. f, Time course of PhPINK1 and TcPINK1 disulphide formation upon treatment with 2 mM H₂O₂, resolved on a non-reducing SDS–PAGE gel. The dimer/oligomer stabilizing PhPINK1 D357A mutation (Fig. 3) enables fast disulphide formation that is averted by an additional C169A mutation. TcPINK1 WT or D359A (equivalent to PhPINK1(D357A)) mutant do not show fast cross-linking behaviour observed in PhPINK1. Engineering of an additional Thr172 to Cys mutation (TcPINK1(T172C/D359A)) results in rapid emergence of cross-linked TcPINK1 dimers upon oxidation. Experiments were performed in biological triplicate with identical results. See Supplementary Fig. 1 for uncropped gels. g, EOPD mutations in the activation segment and αEF–αF loop. Mutations according to¹⁰. h, HsPINK1 EOPD mutants listed in g were expressed in HeLa PINK1^−/− cells, stabilized with OA and treated with H₂O₂ to assess PINK1 dimerization, autophosphorylation and ubiquitin phosphorylation activity (see Methods). The control D384A mutant (mutation of the DFG motif, equivalent to D357A in PhPINK1) was included as an inactive mutant. As anticipated, it is able to dimerize (oxidative cross-link formed) but unable to autophosphorylate or generate phosphorylated ubiquitin. Oxidative dimerization enables separation of pure catalytic mutants and dimerization deficient mutants. Experiments were performed in biological triplicate with identical results. See Supplementary Fig. 1 for uncropped blots.