Substitution of PINK1 Gly411 modulates substrate receptivity and turnover

ABSTRACT The ubiquitin (Ub) kinase-ligase pair PINK1-PRKN mediates the degradation of damaged mitochondria by macroautophagy/autophagy (mitophagy). PINK1 surveils mitochondria and upon stress accumulates on the mitochondrial surface where it phosphorylates serine 65 of Ub to activate PRKN and to drive mitochondrial turnover. While loss of either PINK1 or PRKN is genetically linked to Parkinson disease (PD) and activating the pathway seems to have great therapeutic potential, there is no formal proof that stimulation of mitophagy is always beneficial. Here we used biochemical and cell biological methods to study single nucleotide variants in the activation loop of PINK1 to modulate the enzymatic function of this kinase. Structural modeling and in vitro kinase assays were used to investigate the molecular mechanism of the PINK1 variants. In contrast to the PD-linked PINK1G411S mutation that diminishes Ub kinase activity, we found that the PINK1G411A variant significantly boosted Ub phosphorylation beyond levels of PINK1 wild type. This resulted in augmented PRKN activation, mitophagy rates and increased viability after mitochondrial stress in midbrain-derived, gene-edited neurons. Mechanistically, the G411A variant stabilizes the kinase fold of PINK1 and transforms Ub to adopt the preferred, C-terminally retracted conformation for improved substrate turnover. In summary, we identify a critical role of residue 411 for substrate receptivity that may now be exploited for drug discovery to increase the enzymatic function of PINK1. The genetic substitution of Gly411 to Ala increases mitophagy and may be useful to confirm neuroprotection in vivo and might serve as a critical positive control during therapeutic development. Abbreviations: ATP: adenosine triphosphate; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; Ub-CR: ubiquitin with C-terminally retracted tail; CTD: C-terminal domain (of PINK1); ELISA: enzyme-linked immunosorbent assay; HCI: high-content imaging; IB: immunoblot; IF: immunofluorescence; NPC: neuronal precursor cells; MDS: molecular dynamics simulation; PD: Parkinson disease; p-S65-Ub: ubiquitin phosphorylated at Ser65; RMSF: root mean scare fluctuation; TOMM: translocase of outer mitochondrial membrane; TVLN: ubiquitin with T66V and L67N mutation, mimics Ub-CR; Ub: ubiquitin; WT: wild-type.


Introduction
PINK1-PRKN mitophagy is a stress-activated pathway that identifies and selectively labels damaged mitochondria for their safe disposal through the autophagy-lysosome system (reviewed in [1]). Failure of this critical quality control mechanism is thought to lead to the buildup of macromolecular damage over time, eventually culminating in cell death. As such, complete loss of either enzyme, PINK1 or PRKN, is invariably linked to earlyonset Parkinson disease (PD). Although dopamine neurons appear exquisitely sensitive to loss of mitophagy, emerging evidence suggest a much broader contribution to various agerelated diseases of the brain and beyond. In line with this notion, PINK1 and PRKN are widely expressed and their reduction in cell and animal models provides a sensitized background to a variety of stressors, while overexpression of particularly PRKN, but also PINK1 [1,2], seems broadly cytoprotective. However, both enzymes are tightly regulated, and it remains uncertain whether their continuous overactivation and the potentially resulting excessive mitophagy is beneficial at all times or can be detrimental under certain circumstances.
The kinase PINK1 acts as a sensor and is continuously imported into healthy mitochondria where it is cleaved, released back to the cytosol, and rapidly degraded. Stress and damage prevent this initial import leaving full-length PINK1 to swiftly accumulate locally in the outer mitochondrial membrane with its kinase domain facing the cytosol. For activation, PINK1 dimerizes into a higher molecular weight complex and autophosphorylates [3][4][5][6] to enable its kinase activity toward ubiquitin (Ub) [7][8][9]. Ub phosphorylated at Ser65 (p-S65-Ub) acts as an allosteric activator of and receptor for the E3 Ub ligase PRKN [10][11][12] providing a highly selective label [13][14][15]. Once recruited to damaged mitochondria, PRKN is also phosphorylated by PINK1 in its Ublike domain [16][17][18][19]. Both phosphorylation events are required to activate PRKN from its cytosolic, auto-inhibited state and to fully unleash its E3 Ub ligase functions on mitochondria. The functions of both enzymes then synergize in a feedforward loop where the products of PRKN activity become the substrates for PINK1, coating damaged mitochondria with p-S65-Ub that then also serves as a mitophagy tag [14,15,20].
Although the human structure has remained elusive, crystal structures of insect orthologs provided first insights into the kinase activity of PINK1 [21][22][23] and recent studies detailed the initial activation process as the first step in the mitophagy cascade [7][8][9]. Despite sequence differences, it has become clear that PINK1 is quite distinctive from other known kinases (reviewed in [24]). Human PINK1 contains three unique insertions in the N-lobe of its kinase domain that seem to play major roles in governing its enzymatic function. Insertion loop 2 is required for PINK1 dimerization which facilitates autophosphorylation in trans within its catalytic loop (S228), and not in the activation loop as the majority of kinases [25]. The resulting conformational changes lead to organization of insertion loop 3 and likely to dissociation of the dimer for activated PINK1 to engage with Ub. Moreover, it has been suggested that the preferred substrate of PINK1 is a minor conformation of Ub (less than 1%) with a C-terminal retracted tail that results in greater exposure and accessibility of the S65 residue [26]. While the PINK1-dependent phosphorylation may also change structure and function of Ub, such a conformational constraint could be rate-limiting, despite the vast abundance of Ub in cells.
Recently we had identified a partial dominant negative effect exerted by a heterozygous PINK1 activation loop mutation [27]. In probing the pathogenic mechanism further, we now report that PINK1 G411S is aberrantly autophosphorylated, which leads to reduced substrate phosphorylation. More importantly, we discovered that PINK1 G411A exhibits strikingly enhanced Ub kinase activity compared to the wild-type (WT) form. This effect was conserved across different cell types and translated into enhanced PRKN activation and increased mitophagy rates under endogenous conditions. Structural computational analyses suggested the induction of a conformational change in Ub when bound to PINK1 G411A resulting in increased receptivity and overall better positioning of the substrate S65. Kinase assays confirmed that in contrast to PINK1 WT, the enzymatic activity was not limited to the C-terminally retracted conformation of Ub, but instead PINK1 G411A was able to phosphorylate the common, extended form of Ub comparably well. Our work provides a genetic proof of concept for enhanced PINK1 activity under endogenous conditions that can potentially guide the development of small molecules changing receptivity and ultimately availability of substrates.

PINK1 G411S is aberrantly autophosphorylated
To follow up on the pathogenic mechanism of the G411S variant, we set out to further characterize and compare PINK1 protein from control and PD patient fibroblasts. PINK1 is cleaved in healthy mitochondria, but upon damage, full-length protein accumulates into a supermolecular complex [3,4,7,9]. Size exclusion chromatography of lysates from challenged fibroblasts confirmed a comparable incorporation of both endogenous WT and PINK1 G411S mutant into a higher molecular weight assembly ( Figure 1A). However, levels of p-S65-Ub remained significantly lower in PINK1 G411S cells compared to WT control fibroblasts ( Figure 1B-D). To analyze PINK1 autophosphorylation, which is known to be critical for enzymatic activity [5,6,17], we prepared Phos-tag gels that retard and thereby separate modified from non-modified species. Surprisingly, we noticed additional phosphorylated PINK1 bands in patient cells with the heterozygous PINK1 G411S variant compared to WT controls, despite comparable levels of total PINK1 protein ( Figure 1B).
Knockdown of endogenous PINK1 in HeLa cells together with low level re-transfection of V5-tagged WT or PINK G411S cDNA further confirmed an increase in PINK1 phosphorylation accompanied by a reduction in Ub substrate phosphorylation ( Figure 1E-F). Phosphatase treatment of the samples led to a complete collapse of the shifted bands, as expected for phosphorylated species ( Figure 1G). Moreover, changing the ratio of co-transfected PINK1 G411S to WT from 0 to 100%, resulted in a gradual increase of PINK1 phosphorylation ( Figure 1H). Introduction of the PINK1 G411S variant into a kinase-dead background revealed that PINK1 itself catalyzes the aberrant phosphorylation ( Figure 1I). However, further efforts to determine the mode of the autophosphorylation (in cis or in trans in a dimer) remained inconclusive perhaps due to incompatibility of different epitope tags needed for such experiment (data not shown). Lastly, mass spectrometry of V5-tagged PINK1 immuno-purified from HEK293E PINK1 knockout (KO) cells confirmed that the activation loop mutation Ser411 itself was indeed phosphorylated ( Figure 1J-K).
Taken together, while PINK1 G411S was stabilized and incorporated into the supermolecular complex similar to WT protein, it showed reduced Ub substrate phosphorylation, but was additionally autophosphorylated at the mutation site.

PINK1 G411A is a more active Ub kinase
Most kinases self-activate by phosphorylation within their activation segment which typically stabilizes this region and imposes conformational changes that promote activity [25]. However, the PINK1 G411S mutation introduces a novel autophosphorylation site into the activation loop that diminishes PINK1 kinase activity toward its substrate Ub. In stark    Figure 1. PINK1 G411S results in reduced substrate phosphorylation, but increased auto-phosphorylation. (A) Immunoblot analysis of gel filtration (GF) chromatography fractions collected from human wild type (WT) PINK1 and PINK1 G411S heterozygous (HET) fibroblast lysates. Molecular weight standards of the GF are indicated on top. Shown is the distribution of full-length PINK1 (indicated by arrow) after treatment with valinomycin (VM) for 16 h. Total cell lysates (L) were run in parallel to pooled (top) or individual GF fractions (bottom). VDAC1 and MFN2 served as controls. (B) Immunoblot analysis of human fibroblasts treated with VM over time. Total lysates were separated by conventional or Phos-tag SDS-PAGE to resolve un-/phosphorylated PINK1 (black arrowhead points to the aberrant autophosphorylation of PINK1 G411S ). (C) Total PINK1 and (D) p-S65-Ub levels were quantified by densitometry and normalized to GAPDH as loading control. (E) HeLa cells were transfected with siRNA to silence endogenous PINK1 and with empty vector (EV) or siRNA-resistant V5-tagged WT or PINK1 G411S cDNA. Cells were treated with CCCP for indicated times and lysates were analyzed by conventional and Phos-tag immunoblots. (F) p-S65-Ub levels were quantified by densitometry relative to GAPDH. (G) HeLa cells were transfected with PINK1 siRNA and WT PINK1or PINK1 G411S V5-tagged constructs and treated with CCCP for 3 h. Incubation of samples with alkaline phosphatase (FastAP) resulted in collapse of the bands separated by Phos-tag. (H) Different amounts of V5-tagged WT and PINK1 G411S cDNA were mixed and transfected as indicated, and cells were treated with CCCP for 3 h. (I) WT or PINK1 G411S constructs with or without additional kinase-dead (KD) mutation were transfected and cells were treated with CCCP for 3 h. (J-K) Immunoprecipitated V5-tagged WT and PINK1 G411S were analyzed by mass spectrometry. Shown are spectra of the 4-h treated samples (J) as well as the abundance of unmodified and Ser411 phosphorylated peptide 408-420 in log scale (K). (C, D, F) Shown is the normalized mean ± SD from three independent experiments. Statistical significance was assessed with two-way ANOVA and Sidak's post hoc test (* p < 0.05, ** p < 0.005, *** p < 0.0005).
contrast, introduction of a non-phosphorylatable PINK1 G411A variant resulted in significantly higher levels of Ub phosphorylation compared to PINK1 WT (Figure 2A-B). Puzzled by these findings, we systematically introduced other amino acids at position 411 and tested their activity in CCCP treated cells in comparison to PINK1 WT (Fig. S1). p-S65-Ub levels increased when G411 was substituted with a short nonpolar amino acid such as alanine or valine but decreased then with increasing size of the nonpolar side chain (Fig. S1A). Polar substitutions such as PINK1 G411S all significantly reduced PINK1 activity with longer side chains having a more drastic effect (Fig. S1B). Charged amino acids at position 411 such as aspartic and glutamic acid that sometimes can serve as phospho-mimetic mutants almost completely abrogated p-S65-Ub levels (Fig. S1C). Of all substitutions tested PINK1 G411A was the most active, whereas the PD mutation PINK1 G411S and even more so its phospho-mimetic form negatively impact Ub kinase function.
In order to characterize the effects of PINK1 G411A and compared to PINK1 G411S in more detail, we next performed highcontent imaging (HCI) of PINK1-V5 and p-S65-Ub immunofluorescence staining on the single cell level over time ( Figure 2C). While the average V5 expression levels were similar for all variants, PINK1 G411S showed significantly lower and PINK1 G411A significantly higher levels of p-S65-Ub compared to PINK1 WT ( Figure 2D-E). To directly assess enzymatic activities of the PINK1 variants we performed in vitro kinase assays with PINK1-V5 immunoprecipitated from cells ( Figure 2F-G). Incubation of PINK1-V5 immunoprecipitates with recombinant monomeric Ub confirmed that the substrate kinase activity of PINK1 G411S was significantly lower, while that of PINK1 G411A was significantly higher compared to PINK1 WT. To determine effects on the pathway further downstream, we performed additional experiments using well-established cell models and paradigms [28]. First, we analyzed Ub-charging of PRKN using its active site mutant PRKN C431S that forms a stable oxyester bond with Ub upon activation of the E3 ligase ( Figure 2H-I). Indeed, PINK1 G411S showed significantly reduced, while PINK1 G411A showed significantly increased Ub-charging of PRKN in comparison to PINK1 WT. As an additional readout for PRKN activation, we assessed its mitochondrial translocation by HCI ( Figure 2J-K). Consistently, and compared with WT, PINK1 G411S impaired recruitment to damaged mitochondria, whereas the hyperactive PINK1 G411A variant increased translocation and activation of the E3 Ub ligase PRKN.
To test potential mechanisms of PINK1 G411A and PINK1 G411S , we investigated the autophosphorylation of PINK1 at S228, which plays a crucial role in enabling the Ub kinase activity of PINK1 [7,9]. We used HEK293E PINK1 KO cells to express and immunoprecipitate WT and mutant PINK1-V5 after CCCP treatment. A phospho-specific antibody showed that all three proteins were strongly phosphorylated at S228 and the ratio of autophosphorylated to total PINK1 was similar between WT, PINK1 G411A , and PINK1 G411S ( Figure 2L-M). Mass spectrometry confirmed these results, as the abundance of the p-S228 phosphorylated peptide was similar among all PINK1-V5 immunoprecipitates (Fig. S2). This suggests an effect of residue 411 downstream of PINK1 activation. As an additional readout for PINK1 activity we monitored the phosphorylation of RAB8A at Ser111 [29]. Endogenous RAB8A was successfully immunoprecipitated from PINK1-V5 transfected HEK293E PINK1 KO cells. While the extent of p-S111-RAB8A to total RAB8A was similar between WT and PINK1 G411A at the analyzed time point, PINK1 G411S showed strongly reduced phosphorylation of this indirect PINK1 substrate ( Figure 2L-M).
In summary, while PINK1 G411S and its aberrant autophosphorylation reduced Ub kinase activity and RAB8A phosphorylation, PINK1 G411A strongly enhanced Ub phosphorylation with sustained effects on PRKN activation and recruitment to damaged mitochondria. Of note, for both mutations our data suggest mechanisms independent of PINK1 stabilization on mitochondria and autophosphorylation at S228.

Residue 411 of PINK1 modifies kinase activity under endogenous conditions
To further confirm our findings and the critical role of residue 411 on the endogenous protein level, we introduced the PINK1 G411S and PINK1 G411A variants into the genome of HEK293T cells using CRISPR/Cas9. Upon CCCP treatment, PINK1 protein stabilized swiftly and to a similar extent across all three genotypes ( Figure 3A-B). Phos-tag immunoblots confirmed the additional phosphorylated band specific to PINK1 G411S , while autophosphorylation of WT and PINK1 G411A was comparable. P-S65-Ub was induced earlier in PINK1 G411A cells and levels remained significantly higher throughout the time course, while levels were consistently lower in PINK1 G411S cells compared to PINK1 WT ( Figure 3A, and C). Greater and earlier p-S65-Ub induction in PINK1 G411A cells seemed to be accompanied by degradation of the PRKN substrate MFN2.
Immunofluorescence analysis further confirmed the much stronger p-S65-Ub signal on damaged mitochondria in PINK1 G411A cells, and the decreased accumulation observed in PINK1 G411S cells compared to WT controls ( Figure 3D). We also employed a Meso Scale Discovery sandwich ELISA [30] to quantify the relative amounts of p-S65-Ub upon mitochondrial depolarization ( Figure 3E). In cells encoding PINK1 G411A , p-S65-Ub levels were about 2-3 times higher upon CCCP treatment, while PINK1 G411S cells showed a modest but consistent and significant reduction over time compared to WT. As an alternative readout, we analyzed mitochondrial fractions in a targeted proteomics approach for an absolute quantification (AQUA) of total Ub and the percentage of p-S65-Ub on mitochondria [31] (Figure 3F-G). While there was no difference in absolute Ub levels on mitochondria before depolarization, we found a substantial increase of especially K48-linked Ub over time in the presence of PINK1 G411A . We were also able to confirm the increased levels of p-S65-Ub on mitochondria in PINK1 G411A cells, both of which were consistent with an enhanced activation of the PINK1-PRKN mitophagy pathway.
To ascertain the "pure" PINK1 kinase effect of residue 411, we sought to uncouple the known feedforward loop between both enzymes through which PRKN can amplify p-S65-Ub  [20]. We knocked out PRKN in all three cells lines and compared them back to the parental WT, PINK G411A , or PINK1 G411S cells with endogenous expression of the E3 Ub ligase ( Figure 3H). In line with rather low expression levels in HEK293T cells, deletion of PRKN only moderately reduced the appearance of p-S65-Ub upon CCCP treatment. Yet the observed differences between the three PINK1 genotypes persisted, suggesting that the respective p-S65-Ub signals are produced largely by PINK1 alone, independent of PRKN ( Figure 3I). Altogether, we were able to confirm our findings also on the endogenous level in support of a direct effect caused by a single amino acid exchange in the activation loop of PINK1. Our analyses further suggest a mechanism modifying the Ub kinase downstream of the initial PINK1 activation and independent of PRKN.

Enhanced Ub phosphorylation through PINK1 G411A in midbrain-derived neurons
Encouraged by the striking biochemical phenotype of the PINK1 G411A variant in HEK293T cells, we sought to confirm the phenotype in a biologically relevant model. We selected ReN cell VM, a human neural progenitor cell (NPC) line that can be terminally differentiated into dopaminergic neurons [32,33] (Fig. S3A-B). Side-by-side comparison of undifferentiated NPCs and differentiated neurons treated with CCCP revealed a quite striking difference in protein levels of PRKN, but similar amounts of PINK1 (Fig. S3C-D). While phosphorylation of Ub was detectable in NPCs upon mitochondrial damage, much greater levels of p-S65-Ub were induced in differentiated neurons, most likely due to the higher expression of PRKN. This was also reflected by a larger decline in levels of mitochondrial PRKN substrates over time in differentiated neurons.
Using CRISPR-Cas9, we introduced a homozygous PINK1 G411A or a heterozygous PINK1 G411S mutation into the genome of ReN cell VM NPCs (Fig. S3E). Successful gene-editing and absence of off-target modifications was confirmed by Sanger sequencing (Fig. S3F). For proof of concept, cell clones were immediately analyzed for levels of PINK1 and p-S65-Ub. As expected, introduction of the PINK1 G411A mutation resulted in a robust increase of p-S65-Ub levels even in undifferentiated NPCs with relatively low PRKN expression (Fig. S3G). Consistent with observations from the various non-neuronal cells, introduction of a single PINK1 G411S mutation noticeably reduced p-S65-Ub levels in differentiated neurons (Fig. S3H). Further no viability issues were noted in undifferentiated NPCs or differentiated neurons and the latter were consistently stained with the neuronal marker MAP2. There were also no obvious effects on differentiation or neurite outgrowth under basal conditions. Likewise, mitochondrial and lysosomal staining did not show overt differences between the genotypes ( Figure 4A). A detailed analysis of differentiated neurons by immunoblot confirmed a significant increase of p-S65-Ub for PINK1 G411A , with no change in PINK1 or PRKN levels in these cells compared to WT ( Figure 4B-C). Data obtained with a sensitive p-S65-Ub Meso Scale Discovery ELISA [30] further suggested that p-S65-Ub levels were slightly, but significantly elevated in PINK1 G411A even at baseline in untreated neurons ( Figure 4D). To determine mitophagy rates in these neurons, we employed flow cytometry as a highly reproducible and sensitive method to quantify a large number of cells. Careful gating of single and live cells was followed by analysis of the lysosomal (acidic) to mitochondrial (neutral) Keima signal for each individual cell of at least 20,000 cells per experiment ( Figure 4E-F). After 24 h CCCP treatment, there was a greater induction of mitophagy in PINK1 G411A neurons compared to WT ( Figure 4G). In addition, of the neurons incubated with the depolarizer, those expressing PINK1 G411A showed overall an increased viability ( Figure 4H) pointing to a potential neuroprotective effect exerted by this more active PINK1 variant.
Together our findings confirm the strongly enhanced Ub kinase activity of PINK1 G411A under endogenous conditions and in the context of disease-relevant dopamine neurons. Moreover, our data emphasize the critical role of PRKN in further amplification of the p-S65-Ub signal and the resulting increased turnover of damaged mitochondria that seems to provide some level of resilience to neurons.

Model of the human PINK1 suggests residue 411 influences substrate receptivity and S65 positioning of Ub
To shed more light onto the role of residue 411 and potential mechanisms for modified Ub phosphorylation, we turned to computational modeling. Incorporating recent structural information from insect PINK1 orthologs [21][22][23] we improved our earlier model of human PINK1 protein [27] ( Figure 5A-B). We phosphorylated the critical Ser228 residue in silico and focused on the soluble fragment of PINK1 containing the activated Ub kinase (residues 156-581). A detailed domain-by-domain analysis of the various structures can be found in Fig. S4. To assess substrate binding and positioning, we docked Ub to the autophosphorylated kinase domain of human PINK1 ( Figure 5C-E) resulting in similar orientation and distances regarding N-lobe, insertion loop 3, and activation segment and also matching some of the reported interactions of Ub and insect PINK1 [23]. We then created and compared models for each PINK1 variant: WT (G411), PINK1 G411A , PINK1 G411S , and PINK1 p-S411 , with the additional autophosphorylation in the activation segment. It has been shown that the represent the normalized mean ± SD of four independent experiments. (L, M) V5-tagged PINK1 WT, PINK1 G411A or PINK1 G411S were transfected into PINK1 KO HEK293E cells. PINK1 was immunoprecipitated with V5 and RAB8A with RAB8A antibodies. Precipitates were loaded onto a gel and immunoblots were probed with phospho-specific and total antibodies for PINK1 and RAB8A, respectively. Three independent experiments were quantified. Statistical significance was assessed with one-way (B, G, M) or two-way (D, E, I, J) ANOVA and Tukey's post hoc test (* p < 0.05, ** p < 0.005, *** p < 0.0005). Asterisks on top of data points indicate statistical difference to WT PINK1, while comparison between PINK1 G411A and PINK1 G411S is indicated by brackets.   preferred substrate of PINK1 is not the common conformation of Ub (PDB: 1UBQ), but a minor (<1%), C-terminally retracted form (CR) with an extended S65 containing loop (PDB: 6EQI) [23,26]. Given this selectivity, we docked different Ub substrates including the common Ub and the minor Ub-CR conformation as well as their phosphorylated counterparts (p-S65) to each of the PINK1 variant models. An overview of the 16 different substrate-enzyme complexes can be found in Fig. S5.
For direct comparison, we first overlaid regions containing the activation segments of the four PINK1 variants (residues 388-431) and determined the orientation of the substrate S65 residues with regard to ATP ( Figure 5F). As expected, PINK1 WT showed an improved positioning of the target S65 of Ub-CR compared to regular Ub, while the two phosphorylated versions were less well-aligned. Of note, PINK1 G411A revealed an identical position of the S65 oxygen atom for both Ub and Ub-CR. In contrast, for PINK1 G411S , S65 of Ub was less well- aligned with ATP and the entire activation loop seemed to retract even further in the PINK1 p-S411 model likely to the negative charge of the phosphorylation therein. Changing the perspective, we overlaid all four Ub substrates and looked at the relative position of the PINK1 activation segments containing either G411, G411A, G411S, or p-S411 ( Figure 5G). We observed that upon docking of Ub to PINK1 G411A there was more overall surface-to-surface contact compared to WT, while both PINK1 G411S and its phosphorylated form had less interaction with Ub. Yet, for Ub-CR, the activation loops of all four PINK1 variants were almost entirely matching. It appeared that this minor Ub conformation induced rigidity of the PINK1 activation loop, which in turn might help improve binding. In contrast, both phosphorylated Ub substrates were shifted sideways and less well aligned between the activation loop and ATP. These observations were further supported by calculations of free energy perturbation (FEP+), proximity, and interface areas between the PINK1 variants and the respective Ub substrates (Table S1).
Taken together, PINK1 G411A showed a more favorable alignment for S65 from the common Ub structure suggesting that the mutant kinase may accept both substrate conformations similarly well.

G411A stabilizes the PINK1 structure and induces the minor conformation of Ub
To characterize potential structural changes over time, we performed relatively long (1.5 μsec) molecular dynamics simulations (MDS) of WT and PINK1 G411A , each in complex with ATP and normal Ub (i.e., the common conformation). Root mean square deviations (RMSD) of atomic positions across the duration of the simulation were only subtle for all structures (Fig. S6A-B). We first focused on intrinsic motions in PINK1 (both globally and locally), starting with an analysis of the root mean square fluctuations (RMSF) per residue over time ( Figure 6A). Differences between WT and PINK1 G411A were notable in the N-lobe, where fluctuations seemed especially prominent within the unique insertion loops 1 and 2, with no major changes seen in loop 3. Additional differences between both structures were found in the CTD region. Except for residues from insertion loop 1, the spatial extent of random, uncoordinated motion was generally reduced in PINK1 G411A compared to WT. We next investigated correlated motions based on mass-weighted, normalized covariance matrices. Interestingly, the PINK1 G411A variant seemed to promote coordination between N-and C-lobes of the kinase domain as well as within the C-lobe itself ( Figure 6B-C). Finally, we inspected the secondary structure with a particular focus on the region surrounding residue 411, which exists as either a turn or a 3-10 helix for WT PINK1 (80.98% vs. 19.02% of the time, respectively). ( Figure 6D). For PINK1 G411A , the turn was further increased while the 3-10 helix was reduced (98.18% vs. 1.82% of the time, respectively), which may allow for more regular propagation of motion within and between the two lobes of the kinase domain. However, the number of H-bonds formed between loop 3 and Ub over time was greater for PINK1 G411A than WT ( Figure 6E) as was the buried surface area of the PINK1:Ub complex ( Figure 6F) both of which suggest more favorable interaction between kinase and substrate.
We next turned our attention to the conformational changes in Ub that was bound to each PINK1 model during the simulations. Analysis of the RMSF per residue of Ub revealed distinct peaks ( Figure 7A) that partially overlapped with important motifs and interaction surfaces of Ub [34]. Compared to PINK1 G411A , Ub bound to WT PINK1 showed much greater fluctuations in a region spanning residues 6-13 (peak at Gly10) as well as of the C-terminal tail (aa [73][74][75][76]. In contrast, two other, narrower regions peaking at Asp52 and strikingly at Ser65, were more pronounced in Ub bound to PINK1 G411A compared to WT. Correlated motions of all Ub residues with position 411 of PINK1 during the simulation ( Figure 7B-C) showed only for WT PINK1 a significant positive correlation with Ub residues immediately surrounding Ser65. A closer look at the secondary structure of Ub and especially at residues 73-76 revealed a turnover 35.14% of simulation time in the context of WT PINK1 (Fig. S6C). However, this increased to 79.41% of simulation time when bound to PINK1 G411A , further supporting the overall restricted motions and a greatly reduced flexibility of the C terminus of Ub.
Through additional analyses we sought to determine whether these changes were reminiscent of the minor Ub conformation that seems to be the preferred PINK1 substrate [23,26]. First, the Ramachandran angles adopted by residue Gly10, confirmed a greater stability in that region for Ub bound to PINK1 G411A with a distribution more similar to the Ub-CR structure ( Figure 7D). Second, solvent accessible surface area (SASA) showed a persistently increased exposure of Ser65 when Ub was bound to PINK1 G411A ( Figure 7E). Third, as a measure of C-terminal retraction, the distance between Ala46 and Gly76 showed relatively large oscillations for Ub when bound to WT PINK1, but this was reduced in the context of PINK1 G411A and remained stable for most of the time (last 2/3 of the simulation) ( Figure 7F). Last, we categorized all frames (250 psec/frame) of the simulations into distinct conformational clusters and then analyzed and directly compared them back to the reference Ub structures ( Figure 7G). Ub not only adopted fewer conformations when bound to PINK1 G411A , but the majority of these trajectories fell into structural clusters closely resembling Ub-CR. Weighted energetics calculations confirmed an overall more favorable interaction with the respective Ub conformations in case of PINK1 G411A (−0.55 kcal/mol) compared to WT PINK1 (1.95 kcal/mol). Raw and weighted ΔG values of interaction between PINK1 and Ub, complex stability, and total potential energy for each cluster can be found in Table S2.
Altogether our simulations and comprehensive analyses support a less dynamic PINK1 G411A complex that propagates motions to Ub, which leads to a higher frequency in C-terminal tail retraction and a solvent exposed Ser65, which may allow more efficient phosphorylation of the substrate.

PINK1 G411A is more receptive to and efficiently phosphorylates also the common Ub conformation
Enthused by the structural prediction, we turned to our geneedited cell models again to validate the proposed mechanism by probing substrate selectivity of WT and the PINK1 G411A mutant in vitro. The field struggles to purify recombinant, active human PINK1, so we isolated mitochondria from CCCP-treated cells of different PINK1 genotypes as a source of the human kinase. As further controls, we used mitochondrial preparations from cells that had been left untreated (i.e. without PINK1). As a defined substrate for these in organello kinase assays, we used recombinant monomeric (FLAG-tagged) Ub for different reaction times. With this set up, we were able to confirm the significantly enhanced (G) Ub conformations were sorted using the Quality Threshold algorithm with heuristically determined RMSD cutoffs to obtain a reasonable number of total clusters, with minimum cluster size approximately 1% of the frames, and rejection rate less than 5%. Shown is an overlay of the center frames of the main clusters for WT PINK1 (left) and PINK1 G411A (right) together with the common Ub conformation (1UBQ) in black and the minor Ub-CR structure (6EQI) in gray. Ser65 is shown in Ub kinase activity of PINK1 G411A compared to WT, and the reduced activity of the G411S mutant ( Figure 8A-B), similar to our findings in cells in culture. Next, we sought to corroborate that the C-terminally retracted Ub conformation, which seems to bind PINK1 with higher affinity, is indeed the preferred substrate of the kinase as previously reported [23,26]. For this we used recombinant Tribolium castaneum TcPINK1 protein (which in contrast to human PINK1 can be purified) and directly compared phosphorylation of WT Ub to a Ub variant that adopts the minor, C-terminally retracted conformation regardless of phosphorylation status. This is achieved by introduction of two point mutations (T66V and L67N) and is hereafter referred to as Ub[TVLN] [26] (Figure 8C). Having established a striking difference in phosphorylation of the two substrates, we wanted to determine whether human WT PINK1 or the PINK1 G411A variant similarly discriminate between the common and minor Ub conformation. We thus performed side-by-side in vitro assays with isolated mitochondria containing human PINK1 and either WT Ub or Ub[TVLN] as the substrates ( Figure 8D). Consistent with the structural modeling, PINK1 G411A seemed equally receptive to either Ub substrate and effectively and comparably phosphorylated both ( Figure 8E). WT PINK1 was overall less active than PINK1 G411A but especially less efficient in phosphorylating Ub WT.
In summary, we provide experimental evidence that compared to WT, PINK1 G411A is more receptive to and allows efficient phosphorylation of also Ub WT similarly to the C-terminally retracted mutant. This is consistent with the idea from structural prediction that the PINK1 variant may transform the common Ub structure into the more suitable Ub-CR conformation, thereby increasing the abundance of its preferred substrate conformation which ultimately leads to increased turnover. ball and stick model for these structures as a reference point. The center frame of each unique cluster was subjected to energetics calculation using FoldX and the raw score was weighted by the percentage of frames represented by that particular cluster. The color-matched pie chart depicts the frequencies of each cluster. The respective raw and weighted ΔG values of the interaction between PINK1 and Ub from each cluster can be found in Table S2.

Discussion
The PINK1-PRKN directed mitophagy pathway is emerging as a promising future therapeutic avenue with potentially farreaching implications. The vital roles of both enzymes are well supported by human genetic findings and the broad exacerbating phenotypes in loss of function models. However, direct experimental evidence for their protective effects is scarce and mostly relies on overexpression of PINK1 or PRKN. Likewise, though widely assumed, clear-cut evidence for the beneficial effect of increasing mitophagy is lacking thus far although there are increasing efforts to activate (or derepress) the pathway for potential therapeutic targeting of PD (reviewed in [35]). By probing further into the mechanisms of the pathogenic PINK1 G411S mutation, we herein identify a novel, potentially protective variant with strikingly enhanced enzymatic activity compared to WT PINK1. Based on structure-function analyses we propose that PINK1 G411A effectively changes Ub receptivity and positioning, and thus expands the actual abundance of the minor conformation of Ub that is the preferred substrate. The resulting increase in Ub phosphorylation is robust and consistently seen across different models and under endogenous conditions in gene-edited cells. Increased levels of p-S65-Ub translate further into augmented PRKN activation and greater mitophagy rates that seem to protect differentiated neurons in culture from mitochondrial stress. Our genetic proof of concept model will lead to a better understanding of the consequences of boosting PINK1-PRKN mitophagy under endogenous conditions. The PINK1 G411A variant may not only be useful as a critical positive control but may also help guide the development of small molecule PINK1 activators.
While complete loss of PINK1 or PRKN invariably results in early-onset PD, the contributions of reduced function to disease later in life remains uncertain. The most recent largescale genetic studies failed to provide unequivocal support for a role of heterozygous mutations as a robust risk factor for PD [36][37][38]. However, structural and functional analyses have clearly demonstrated that not all mutations are alike. In fact, much has been learned about the regulation and sequence of events along the pathway from missense variants with distinct biochemical or cellular phenotypes [39][40][41][42][43][44][45]. Our previous work revealed a sizable contribution of the PINK1 G411S mutation to risk and onset of PD caused by a distinctive partial dominant-negative mechanism of the mutation [27,46]. Probing further into the mechanisms of PINK1 G411S using primary patient and gene-edited cells, we here confirm and expand our earlier findings. We demonstrate that PINK1 G411S is indistinguishable from WT with regard to its stabilization and dimerization into a supermolecular complex upon mitochondrial stress. Yet, we also uncover that PINK1 G411S hyper-autophosphorylates at the newly introduced serine residue in the activation loop, which seems to repel the Ub substrate. While most kinases are activated by autophosphorylation in that region [25], PINK1 autophosphorylates at S228 in the catalytic loop which is critical for activation and enables recognition and binding of Ub [3][4][5][6][7]9].
More importantly though, we also identify a more active form of PINK1 here during our investigation. When introducing G411A as a variant, the resulting p-S65-Ub levels were significantly elevated in comparison to WT PINK1, and this was consistently seen across cell types and under endogenous conditions. Despite a potential involvement of G411 in dimerization of PINK1 [7,9], it is noteworthy that we found no evidence that mutations of this residue would alter stabilization or autophosphorylation of PINK1 at S228. Rather our data collectively points to an effect downstream of the initial PINK1 activation consistent with the increased substrate receptivity caused by the transformation of the Ub structure when bound to PINK1 G411A . Overall we propose that PINK1 G411A stabilizes the kinase fold causing Ub to more often adopt the minor, C-terminally retracted conformation, which better exposes Ser65, thereby effectively increasing the abundance of this preferred PINK1 substrate. However, it needs to be clarified, whether the PINK1 G411A variant is indeed causing a more effective conversion of the structure of Ub or bypassing the need for this conformational change.
Notwithstanding, what has already become clear is that small upstream effects are amplified along the pathway through the concerted actions of the Ub kinase-ligase pair. While the feedforward loop in which PRKN provides more substrates for PINK1 resulting in further PRKN activation and recruitment is well known, our data further emphasize this could indeed be exploited for therapeutic intervention. When knocking out PRKN in HEK293T cells gene-edited for PINK1 G411A , p-S65-Ub level were only moderately reduced, consistent with the relatively low endogenous expression of the E3 Ub ligase. Conversely, upregulation of PRKN during differentiation of gene-edited ReN cells from precursor to neurons boosted the p-S65-Ub signal. While precursor and neurons both showed comparable PINK1 protein levels and stabilization over time upon depolarizer treatment, PRKN expression was about 20-fold higher resulting in an about 3-4 fold increase of p-S65-Ub. However, whether PINK1 G411A also directly enhances the phosphorylation of Ser65 in the Ublike domain of PRKN, similar to Ub, cannot be clarified in cell models alone as elevated levels of p-S65-Ub also indirectly lead to increased PRKN activation and phosphorylation by PINK1. Regardless, small molecules mimicking the described G411A effect may be useful to also compensate for reduced expression or enzymatic activity of either PINK1 or PRKN to boost mitophagy, for instance in heterozygous mutation carrier at risk for developing disease.
Nevertheless, our study also has clear limitations. Most importantly, and although in excellent agreement with the published insect structures, several of our findings are based on computational predictions of human PINK1. Yet, we performed relatively long molecular dynamics simulations (~1.5 µsec) and do provide experimental data supporting the main conclusion -PINK1 G411A can phosphorylate Ub WT (i.e., the common conformation) similarly well to the Ub[TVLN] mutant (reflecting the minor, C-terminally retracted conformation) that is the more suitable substrate for WT PINK1. However, given that human PINK1 has been notoriously difficult to purify, our kinase assays were performed in organello -using purified mitochondria from gene-edited cells as the source for human PINK1 -and not reconstituted in vitro with pure, recombinant proteins. Future advancements in expression and purification methods should provide larger quantities of human PINK1 needed for critical evaluation and definitive proof of the proposed mechanism. The following experimental structural analyses should be able to inform about Ub positioning and conformation. In this context it might be informative to include additional PINK1 substrates, such as PRKN as well as poly-Ub chains of different lengths and linkages that are "free" or conjugated to a mitochondrial substrate protein of PRKN. Additional biophysical experiments should help establish binding modes and kinetic profiles in combination with the common or the minor Ub conformation (WT Ub or Ub[TVLN] mutant, respectively).
While we have already shown that substitution of G411 with polar or charged amino acids all lead to reduced p-S65-Ub levels, additional mutation scanning in the PINK1 activation segment around residue 411 of human PINK1 could reveal further insights into this novel mechanism of action. Although G411 is conserved in mammals, it is quite divergent in lower organisms such as insects, worms and zebrafish. Notably, C. elegans and D. melanogaster both carry an alanine, while zebrafish has a serine at position 411. Given these sequence differences it could be interesting to compare PINK1 from these species to human in order to establish a structure-activity relationship also with respect to dimerization and Ub binding. These studies might enable future drug design, in addition to but different from the currently studied ATP analogs [47,48], capitalizing on the transformational changes in Ub receptivity and positioning seen for PINK1 G411A .
Lastly, while we do provide a genetic model for PINK1 activation, the full consequences and thus potential therapeutic value of enhancing Ub phosphorylation under these endogenous conditions remain to be established. In general support of a beneficial role, we do find a promising increase in mitophagy rates and a concomitant protection of geneedited neurons upon mitochondrial stress. Yet, to highlight long-term beneficial effects and/or potential issues arising from a more active form of PINK1, it will be essential to carefully characterize mitochondrial health and cellular fitness at both baseline and over time under chronic or acute stress conditions. Beyond the current cell models, studies should extend to more complex and disease-relevant iPSC-derived culture systems. Unbiased investigations of transcriptome, proteome, and metabolome should help flag any obscure alterations. Animal work is eventually needed to exclude potential concerns in the organismal context, within the brain and in peripheral tissues in which PINK1 and PRKN have been shown to play a role [49]. Studies during development and normal aging as well as under different disease conditions should further help de-risk pharmacological efforts and provide additional support for boosting PINK1-PRKN mitophagy as viable therapeutic avenue.

Chemical treatments and transient siRNA and DNA transfection
Carbonyl cyanide m-chlorophenyl hydrazone (CCCP; Sigma Aldrich, C2759) was used at a concentration of 10 µM for all experiments with HEK293 and HeLa and 20 µM for all ReN cell experiments. Valinomycin (Cayman Chemical, 10009152) was used for experiments with fibroblasts at 1 µM. HeLa cells were plated 20-24 h before transfection with siRNA/DNA using Lipofectamine 2000 (Thermo Fisher Scientific, 11668019). The following amounts were used for transient siRNA and DNA transfection per 6-well: 3 µl of 20 nM siRNA, 500 ng of PINK1 cDNA, and 4.5 µl of Lipofectamine per 250 µl of Opti-MEM media (Thermo Fisher Scientific, 51985034). For HCI, media was replaced after 4 h and cells were cultured for an additional 20 h. For immunoblot analysis, media was replaced after one day. Control siRNA (all stars negative control, Qiagen, 1027281) or PINK1-specific siRNA (5'-GACGCTGTTCCTCGTTATGAA-3', Qiagen) were used along with siRNA-resistant PINK1 cDNA. PINK1 constructs with C-terminal V5 or mCherry tag have been described previously [27]. PINK1 G411 substitutions were introduced by site-directed mutagenesis of WT or KD (kinase dead: K219A+D362A+D384A) PINK1 cDNA constructs and confirmed by sequencing.
For phos-tag gel electrophoresis, freshly prepared samples were separated on 6% (PINK1) or 14% (Ub) Tris-Glycine gels containing 50 µM Phos-tag acrylamide AAL-107 (Wako Chemicals, 304-93521) and 100 µM MnCl 2 . Prior transfer onto PVDF membranes, gels were soaked in ddH 2 O, then washed once for 20 min in transfer buffer containing 10 mM EDTA and 0.01% of SDS and twice in the absence of EDTA. Silver staining was performed according to the manufacturer's instructions (Thermo Fisher Scientific, 24612).

Antibodies
The following antibodies were used for immunoblot (IB) and immunofluorescence (
For PINK1 phosphorylation analyses, samples were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. Bands around 60 kDa were cut out, destained in 50% acetonitrile, 50 mM Tris, pH 8.2 until clear, and then reduced with 25 mM TCEP, 50 mM Tris, pH 8.2 at 60°C for 30 min, followed with alkylation using 10 mM iodoacetamide:50 mM Tris pH 8.2 at room temperature for 30 min in the dark. Proteins were digested in-situ with 0.1 ug trypsin (Promega, VA9000) in 25 mM Tris, pH 8.2, 0.0002% Zwittergent 3-16 (Calbiochem, 693023), at 37°C overnight, followed by peptide extraction with 2% trifluoroacetic acid and acetonitrile. The extracted peptides were analyzed using a Thermo Scientific Eclipse Tribrid mass spectrometer coupled to a Vanguish Neo HPLC system. The digest mixtures were loaded onto a Halo C18 2.7 µm EXP stem trap (Optimize Technologies) and chromatography was performed using 0.1% formic acid in both the A solvent (98% water:2% acetonitrile) and B solvent (80% acetonitrile:10% isopropanol:10% water), with a 3% B to 40% B gradient over 60 min at 350 nl/min through a PepSep C18 2.4 um, 100 um x 40 cm column (Bruker, 1895837). The Eclipse mass spectrometer was set for data dependent acquisition with a 2 second cycle time between the MS1 survey scan from 340-1500 m/z at resolution 120,000 (at 200 m/z), followed by EThcD MS/MS scans at resolution 30,000 with the isolation width set to 1.6 m/z. Ions with charge states of 2 to 4 were allowed for MSMS and, if selected, were placed on an exclusion list for 60s. The normalized AGC target settings were 200% for the MS1 and 200% for MS2 scans with max ion inject times of 50 ms and 54 ms, respectively.
The raw files were processed with Proteome Discoverer (ver. 2.5.0.400), searching with SequestHT against a Swissprot human database that contained the PINK1 variants (2021_01; 8135 entries) with static modifications set for carbamidomethyl cysteine. The variable modifications allowed were phosphorylation at serine, threonine, and tyrosine, and oxidation at methionine. Peptide identifications were filtered at 1% false discovery rate and phospho-site confidence required a probability of 75% or higher.

Analysis of PRKN Ub charging
HeLa 3xFLAG-PRKN C431S control cells and cells transfected with PINK1 siRNA and siRNA-resistant PINK1 were treated with 10 µM CCCP were harvested in preheated (95°C) SDS lysis buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 1% SDS). Lysates were homogenized by 10 strokes with a 20 G needle. To verify the band shift by oxyester formation, aliquots of lysates were treated with or without NaOH (final concentration 100 mM) for 1 h at 37°C before loading onto SDS gels. 3 µg protein were analyzed by IB.

High-content imaging (HCI) and immunostaining of cells
HeLa or HeLa EGFP-PRKN cells transfected with PINK1 siRNA and siRNA-resistant PINK1 cDNA were seeded with 5,000 cells per well in 96-well imaging plates (Fisher Scientific, 08772225). Twenty-four h after plating, cells were treated with 10 µM CCCP, fixed for 10 min with 4% PFA and stained with Hoechst 33,342 (1:5,000: Thermo Fisher Scientific, H21492). HeLa cells were incubated with primary anti-p-S65-Ub and anti-V5 antibodies for 2 h, followed by incubation with secondary antibodies (anti-rabbit IgG Alexa Fluor 488 and anti-mouse IgG Alexa Fluor 568; Thermo Fisher Scientific, A11034, A11004). HCI plates were imaged on a BD Pathway 855 system (BD Biosciences, Franklin Lakes, NJ, USA) with a 20x objective using a 3 × 3 montage (no gaps) with laser autofocus every second frame. Raw images were processed using the builtin AttoVision V1.6 software. Regions of interest (ROIs) were defined as "cytoplasm" and "nucleus" using the built-in "RING 2 outputs" segmentation for the Hoechst channel after applying a shading algorithm [28]. For p-S65-Ub, the cytoplasmic intensity of each cell was normalized to the respective V5 intensity. EGFP-PRKN translocation measurements [28] were restricted to mCherry-positive cells, defined as cells with mCherry signal equal or higher the overall average intensity. ReN cells VM were grown on Matrigel coated coverslips and differentiated. Cells were fixed with 4% paraformaldehyde for 10 minutes before membranes were permeabilized with 1% Triton X-100 in PBS (Boston Bioproducts, BM-220) for 10 min. Cells were stained with TOMM20, LAMP2, and MAP2 antibodies, followed by secondary antibodies (anti-rabbit IgG Alexa Fluor 488, antimouse IgG Alexa Fluor 568, anti-chicken Alexa Fluor 647, Thermo Fisher Scientific, A11034, A11004, A21449). Nuclei were counterstained with Hoechst 33342 (1:5000 in PBS). Representative images were obtained by AxioObserver microscope equipped with an ApoTome Imaging System (Zeiss, Oberkochen, Germany).

CRISPR-Cas9 gene-editing and lentivirus
We used CRISPR-Cas9 to introduce a single nucleotide exchange by replacing the GGC (Gly411) with an AGC (Ser411) or a GCC (Ala411) triplet. HEK293T cells were transfected with 1 µg pX330-sgRNA-Cas9 plasmid (Addgene, 42230; deposited by Feng Zhang) and 10 μg single stranded oligo (Integrated DNA Technologies) using X-tremeGENE 9 (Sigma-Aldrich, 6365787001) according to the manufacturer's instructions. ReN cells VM stably expressing mitoKeima were transfected using the nucleofector P3 kit (Lonza, V4XP-3032). Single cell colonies were generated by limited dilution of transfected cells in 96-well plates. All clones were analyzed by Sanger sequencing. Sequences were analyzed using SnapGene software. In the case of ReN cell VM, we introduced a silent blocking mutation downstream of the intended change that allowed prescreening of clones with a BanI restriction digest. Because of a triplication of chromosome one in HEK293T cells, we further amplified genomic DNA of positive candidates with PCR and cloned the PCR products into a pGEM T Easy vector (Promega, A1360) to confirm that all alleles had been edited correctly. After confirming on-target editing, we also excluded off-targets by Sanger sequencing of candidate regions as identified by the Benchling biology software (2021, www.benchling.com) and confirmed similar expression of PINK1 and PRKN by qRT-PCR and IB. For HEK293T cells, we further monitored cellular ROS (CellRoX), mitochondrial ROS (mitoSOX), mitochondrial membrane potential (JC10), and cellular respiration (Seahorse mitochondrial stress test; Agilent, 103015-100) and found no difference to parental, un-edited cells. Further modification of genome-edited HEK293T cells was performed using lentivirus to knock out PRKN with a sgRNA targeting the start codon of PRKN. Antibiotics were used to select positive cell populations and single clones were analyzed by immunoblot. HEK293E PINK1 KO cells have been described previously [30].

Quantitative mass spectrometry of mitochondria
HEK293T cells expressing WT PINK1, or CRISPR edited to express PINK1 G411A or PINK1 G411S were treated with 10 µM CCCP for the time indicated. Cells were harvested and subjected to crude mitochondrial isolation as described previously [20,31]. Briefly, crude mitochondria samples were prepared by sonication of cells resuspended in mitochondrial isolation buffer ( ) and mitochondria were then isolated by differential centrifugation before lysis. Mitochondrial extracts were sonicated, clarified by centrifugation, and protein concentrations determined by the Bradford assay (Bio-Rad, 5,000,006). Quantitative mass spectrometry was performed on mitochondrial extracts (3 µg) using PRKN target-Parallel Reaction Monitoring (Pt-PRM) platform using an optimized series of heavy reference peptides for PRM analysis of Ub chain linkages, and p-S65-Ub [52] and data were collected on an Orbitrap Fusion Lumos instrument.

Expression and Purification of Ub-WT-6XHistidin and Ub-TVLN-6XHistidin
Human Ub DNA was subcloned into a pET60 vector (Novagen, 71851) for ubiquitin-6His-tag. PCR mutagenesis was used to generate Ub[TVLN]-6His-tag mutant. The corresponding proteins were produced in Rossetta (DE3) E. coli cells grown in 1 L terrific broth (Sigma-Aldrich, T0918). When the OD reached 0.6, the proteins were induced using 1 mM IPTG (Goldbio, I2481C) and further incubated for 21 h at 25°C. The E. coli cells were then collected, resuspended in 40 ml lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.06% BME, 2 tablets of Roche protease inhibitors), and lysed by tip-sonication. After removing the cell debris through ultracentrifugation (20,000 x g, 10 min), the supernatant was filtered through a 0.2-µm filter and incubated with the Ni-NTA beads (Pierce, 88221). After three times of wash, the 6XHis tagged proteins were eluted with Tris-HCl buffer containing 500 mM imidazole. The eluate was then loaded on a gel-filtration Superose 6

Mitochondrial preparations and kinase assays with mitochondria
For fractionation into mitochondrial and cytoplasmic fraction cells were washed scraped in fractionation buffer (20 mM HEPES, pH 7.6, 220 mM mannitol, 10 mM potassium acetate, 70 mM sucrose) containing protease and phosphatase inhibitor cocktails and homogenized with 10 strokes through a 27 G needle. Lysates were spun at 800 x g for 5 min at 4°C to remove nuclei and post-nuclear supernatant was spun at 8000 x g for 20 min at 4°C to pellet mitochondria. Protein concentration was determined using a BCA assay and 50 µg of mitochondria were used per kinase assay reactions. Mitochondria were resuspended in 10 µl of kinase reaction containing 10% ATP regeneration buffer (20 mM HEPES, pH 7.6, 10 mM ATP, 300 mM phosphocreatine [Sigma-Aldrich, P7936], 10 mM MgCl 2 , 10% glycerol, 1.5 mg/ml creatine phosphokinase [Sigma-Aldrich, C3755]), 1 µg of FLAG-or His-tagged Ub (Boston Biochem, U-120, U-530) in fractionation buffer and incubated at 30°C for 2 or 4 h. Mitochondria were spun down for 5 min at 20,000 x g and supernatant was collected and mixed with 6x sample buffer; 10 µl were loaded per lane.

Flow cytometry analysis of mitoKeima
Analysis of mtKeima was performed using an Attune NxT (B/ R/Y/V) flow cytometer (Thermo Fisher Scientific, Waltham, MA, USA). Neutral mtKeima signal was collected with 405 nm excitation and 603/48 nm emission (standard VL3 detector optical path). Acidic mtKeima signal was collected with 561 nm excitation and custom optical settings for the YL2 detector, generated by replacing the 650DLP with the 740DLP filter and removing the BP620/15 filter, resulting in capture of 600-740 nm emission. Height signals were collected and analyzed in log scale, without compensation. Data were analyzed with FCS Express 6 (De Novo Software) using standard doublet discrimination, followed by viability determination with Sytox Red. The resulting single, live cells (at least 20,000 per experiment) were displayed on a bivariate plot of neutral (x-axis) vs. acidic (y-axis) mtKeima signal and the data exported for calculating the ratio of acidic to neutral Keima signal for each cell.

Densitometry and statistical analysis
Densitometric analysis of western blots was performed by using ImageStudio Lite software version 5.2.5. Data analysis and visualization were performed by GraphPad Prism version 8. Statistical comparisons were performed using parametric analysis with either using student's two-sided t-test, one-way and two-way ANOVA with Tukey's post-hoc test (*p < 0.05, **p < 0.005, ***p < 0.0005), depending on the number of groups and experimental design.

PINK1 model construction
The sequence of human PINK1 was taken from the NCBI Reference Sequence NG_008164.1. Amino acid (aa) residues 3-581 were used to generate a complete structural model. The PINK1 sequence was constructed based on multiple alignments, in which each domain was modeled as a separate unit, which were then assembled into a final composite hybrid model. The model was generated from consensus between the programs PRIME (Prime v3.0, Schrodinger, LLC, New York, NY) [53,54], and YASARA SSP/Homology/PSSM Method [55][56][57][58]. The variable loops and gaps were filled using homology and knowledge-based potentials with YASARA. Each missing loop was modeled using the Loop Search module in Sybyl 8.0 or with YASARA loop modeler [55][56][57][58][59][60][61][62]. The selection of final loops was based on highest homology, as well as lowest root mean square deviations (RMSDs). The side chains and rotamers were adjusted with empirical potentials, simulated annealing with explicit solvent, and small equilibration simulations using YASARA's refinement protocol. These were verified by WHAT-IF and PROCHECK [63].
Refinement of the hybrid model was carried out using a limited molecular dynamics-based refinement in YASARA consisting of the Secondary Structure Prediction (SSP) for YASARA parameterization, pKa assignment, solvation and simulated annealing and pre-equilibration setup, i.e. energy minimizations [55,56,59,61]. Both homology and fold recognition were considered, and a final refinement with the entire model was completed using YASARA for 250 psec of molecular dynamics embedded in an empirical force field. The model was then subjected to energy optimization with Polak-Ribiere conjugate gradient (PRCG) with an R-dependent dielectric for finalization.

Molecular dynamics simulation (MDS)
Molecular dynamics was completed on each model for conformational sampling in order to examine any dynamical conformational differences that may occur between the WT or G411A variant. Briefly, each PINK1-Ub system was minimized with relaxed restraints, using steepest descent (SD) PRCG, and equilibrated in solvent with physiological ionic strength [64][65][66][67]. The refinement included the following steps: (1) Energy minimization of explicit water molecules and ions, with protein constrained; (2) Energy minimization of the entire system; (3) MDS for >10 nsec to ensure system acclimatization to the force field (OPLS3/Amber) [68,69]. Succeeding the refinement protocol, 1.5 µsec simulations were completed to collect data for analyses. More complete detail of our minimization/equilibration refinement protocol, as well as simulation can be found in the literature [70][71][72][73].

MDS and modeling analytical methods
Clustering was performed using the Quality Threshold algorithm [74,75] with RMSD cutoff of 1.75 Å (determined heuristically) for both PINK1 WT and G411A. The central structure for each cluster was uploaded to PyMOL and Ub-CR (1ubq.pdb) [76] or Ub-common (6eqi.pdb) [23] crystal structures were aligned to each of them in order to obtain RMSD. FoldX plugin with YASARA [77,78] was used to calculate the dG interaction energy between ubiquitin and PINK1 for each central cluster member, which was then multiplied by the percent of simulation time that conformation was adopted and to generate a weighted dG interaction score to account for conformational propensity.
Post-MDS analyses were performed using VMD [79]. The random rotation/translation was removed via alignment (least squares CONCa backbone superposition onto first frame) as specified in each figure, e.g., ubiquitin chain. RMSD over time was calculated using VMD's RMSD trajectory tool. RMSF was calculated using a custom Tcl/Tk script. Secondary structure per residue per frame was calculated via VMD's Timeline plugin, followed by a custom Visual Basic function to calculate the percent occupancy of specific secondary structure characters. SASA was calculated via VMD's Timeline plugin. Ramachandran distribution plots were generated via VMD's Ramaplot plugin. Hydrogen bonds measurements were taken using the Hydrogen Bonds analysis extension in VMD. The BSA for each frame was computed via a custom Python script measuring and summing the solvent accessible surface area (SASA) of each chain separately, then subtracting the SASA of the complex, then dividing by 2 for correction (SASA PINK1 + SASA Ub -SASA complex)/ 2, where the SASA probe radius is 1.4 Å).
Grcarma [80] was used to generate mass-weighted, normalized covariance matrices. These matrices were then plotted as 2D graphs with respect to residues of interest, as well as appended to the beta column via a custom script and displayed using PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.) All molecular structure graphics were generated with PyMOL.