phospho-Ser65 triggers cleft widening in the N-terminal region.

<p><b>A</b>) Center view of a newly defined pocket in Parkin with Ser65 positioned towards the middle. The cleft is formed between the flexible linker region [<i>cleft wall 1</i>: Arg97, Ser110, Val105, Val111, Leu112, Asp115 Ser116, Val117, and Gly118] and the UBL domain [<i>cleft wall 2</i>: Met1, Ile2, Val3, Phe4, Ser19, Leu61, Asp62, Gln63, Gln64, Ile66, and Val67]. Parkin is rendered in solvent accessible surface colored by atom type (nitrogen-blue, oxygen-red, carbon-cyan). Yellow double-sided arrow indicates cleft regions that were used for center-of-mass calculations: CoM1 (Ser110, Val111, Asp115) to CoM2 (Met1, Ile2, Val3, Phe4, Gln63). The initial cleft width is given for time equal zero. <b>B</b>) The ribbons diagram for Parkin is shown for comparison with same orientation. Relevant domain labels are given. <b>C</b>) Parkin pSer65 is shown after 20 ns of unbiased MDS. The yellow arrow indicates the increased cleft distance. <b>D</b>) Superposition of Parkin structures after 100 ns MDS. Shown are structures for Ser65 and pSer65 as well as for S65A, S65D and S65E variants. Arrows indicate the cleft distances of Ser65 and pSer65. <b>E</b>) Plot for CoM1 to CoM2 distance over time from MDS. Graph shows a relatively closed and stable cleft for unmodified Ser65 (black) of about 8 Å in distance, while pSer65 (green) shows a much more wider cleft from the start, ranging from 11–14 Å over time. Phospho-mimic mutants S65D (magenta) or S65E (blue) as well as the phospho-dead mutant S65A (red) show a strong increase in cleft size over time reaching distance observed with pSer65. <b>F</b>) Solvent-Accessible-Surface-Area (SASA-Ų) within the pocket enclosing Ser65 measured in Ų units. While Ser65 maintain a relatively stable SASA, values for pSer65 strongly increase over time. An increase in SASA over time is also observed for the substitutions S65D and S65E as well as for S65A after an initial decrease. <b>G</b>) Shown are numbers of H₂O molecules within the cavity surrounding Ser65 during MDS. While Ser65 constantly maintains twelve H₂O molecules, pSer65, S65D, S65E, and S65A show a greater solvation of the cavity, consistent with an increased cleft distance and improved SASA.</p

Phosphorylation of Ser65 releases the safety belts of Parkin.

<p><b>A</b>) Zoom into safety belt 1: The UBL blocks RING1 and IBR domains. Key cysteine residues of the E2 binding site in RING1 are indicated. The E2 binding site was defined as follows: Ile236, Thr237, Cys238, Ile239, Thr240, Cys241, Thr242, Asp243, Val244, Arg245, Ile259, Cys260, Leu261, Asp262, Cys263, Phe264, His265, Leu266, and Tyr267 <b>B</b>) The distance between UBL domain (Leu26) and RING1 (Cys238) significantly increased over time MDS. <b>C</b>) Similarly, the distance between UBL (Leu26) and IBR (Phe364) domains significantly increased over time MDS. <b>D</b>) Zoom into safety belt 2: The REP region blocks the E2 binding site in RING1 (as defined in A). <b>E</b>) Dynamic change in REP-RING1 interaction during Parkin opening motion. Graph shows the release of the REP region from the E2-binding site in RING1 as measured by RMSD. The RING1 is released from the REP region by MdMD time of 20 ns, exposing the E2 binding site. <b>F</b>) Loosened interaction between the center Tyr391 in REP region and Cys238 in RING1. The distance increases from 10 to 20 Å. During longer simulations, the distance eventually collapses as the UBL domain moves away and E2 binding has transiently occurred. Across many replicates, we find that the availability of adequate space for an E2 enzyme to approach the binding site in RING1 begins somewhere between 5–22 ns. <b>G</b>) Zoom into safety belt 3: Cys431 is buried by RING0. <b>H</b>) Release of the active site (Cys431) from RING0 (Arg163 C-alpha atom) as measured by RMSD for center-of-mass. RMSD increases moderately over time indicative of a less compacted area. <b>I</b>) SASA for Cys431 entire residue. During MDS, more water is available to Cys431, indicating its enhanced exposure.</p

Protein-protein docking for Parkin and a charged E2∼Ub complex.

<p><b>A</b>) Ten conformations spanning closed Parkin to fully opened Parkin (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003935#pcbi-1003935-g004" target="_blank">Figure 4</a>) were sampled for protein-protein interactions. The E2-Ub complex was docked at the midpoint UBL position (state 2/3) when the REP region liberated the binding site in RING1 (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003935#pcbi-1003935-g004" target="_blank">Figure 4B/C</a>). This conformation showed fewer steric clashes and lowest energy profile. The docking in the same position is shown and rotated 180° to reveal the other side. Residues of the Ubch5a-Ub complex are indicated by color (dark green and brown, respectively). <b>B–D</b>) Docking at the RING1 interface is critical for E2-Ub progression towards the active site of Parkin. E2 binding at RING1 limits the UBL-linker mobility preventing the drift back to the original, auto-inhibited state. <b>B</b>) Same orientation as in A (left side) predicted as an optimal docking conformation after 0 ns of MDS. The distance between Gly76 of Ub and Parkin's active site (Cys431) is indicated. <b>C</b>) Following unbiased MD (>200 ns), the E2-Ub complex moves towards the Parkin's active center. The decreased distance is shown after 50 ns. <b>D</b>) Overall re-orientation of the UBL domain and the E2-Ub complex is shown after 200 ns.</p

Activation, domains, structures and models of human Parkin.

<p><b>A</b>) Schematic representation of Parkin's 2D structure and its activation mode. <i>Top</i>: PINK1-dependent phosphorylation of Ser65 has been shown to activate Parkin. During activation, an Ub-loaded E2 enzyme binds Parkin and catalyzes the Ub transfer onto the active site, Cys431, in order to charge Parkin with the small modifier protein. <i>Bottom</i>: Shown are color-coded functional domains of human Parkin (residues 1–465): Ubiquitin-like (UBL, red), flexible linker (gray), RING0/Unique Parkin domain (R0/UPD, green), RING1 (R1, blue), in-between RING (IBR, purple), Repressor element of Parkin (REP, yellow), RING2 (R2, pink). The putative E2 binding site in RING1 is indicated by a black line. Gray lines indicate the position of the Zn²⁺ coordinating cysteine/histidine residues in the different RING domains. <b>B</b>) Superposition of Parkin's molecular structures. Table lists recently resolved X-ray structures that have been used to generate a model for human full-length Parkin with all-atom resolution. Key residues Ser65 and Cys431 are shown as sticks with carbon in gray, nitrogen in blue, oxygen in red, and sulfur in yellow. <b>C</b>) Molecular modeling of Parkin pSer65. The ribbon diagram for an all-atom molecular structure of Parkin is given, presenting an <i>in silico</i> model of a PINK1-phosphorylated, and thus activated conformation of Parkin. Color matches that of the domain key indicated, and as above. The phosphorylated Ser65 is shown along with the active site Cys431 as Van der Waals (VdW) spheres colored by domain. Zn²⁺ atoms are shown as blue spheres.</p

Age- and disease-dependent increase of the mitophagy marker phospho-ubiquitin in normal aging and Lewy body disease

<p>Although exact causes of Parkinson disease (PD) remain enigmatic, mitochondrial dysfunction is increasingly appreciated as a key determinant of dopaminergic neuron susceptibility in both familial and sporadic PD. Two genes associated with recessive, early-onset PD encode the ubiquitin (Ub) kinase PINK1 and the E3 Ub ligase PRKN/PARK2/Parkin, which together orchestrate a protective mitochondrial quality control (mitoQC) pathway. Upon stress, both enzymes cooperatively identify and decorate damaged mitochondria with phosphorylated poly-Ub (p-S65-Ub) chains. This specific label is subsequently recognized by autophagy receptors that further facilitate mitochondrial degradation in lysosomes (mitophagy). Here, we analyzed human post-mortem brain specimens and identified distinct pools of p-S65-Ub-positive structures that partially colocalized with markers of mitochondria, autophagy, lysosomes and/or granulovacuolar degeneration bodies. We further quantified levels and distribution of the ‘mitophagy tag’ in 2 large cohorts of brain samples from normal aging and Lewy body disease (LBD) cases using unbiased digital pathology. Somatic p-S65-Ub structures independently increased with age and disease in distinct brain regions and enhanced levels in LBD brain were age- and Braak tangle stage-dependent. Additionally, we observed significant correlations of p-S65-Ub with LBs and neurofibrillary tangle levels in disease. The degree of co-existing p-S65-Ub signals and pathological PD hallmarks increased in the pre-mature stage, but decreased in the late stage of LB or tangle aggregation. Altogether, our study provides further evidence for a potential pathogenic overlap among different forms of PD and suggests that p-S65-Ub can serve as a biomarker for mitochondrial damage in aging and disease.</p> <p><b>Abbreviations:</b> BLBD: brainstem predominant Lewy body disease; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DLB: dementia with Lewy bodies; DLBD: diffuse neocortical Lewy body disease; EOPD: early-onset Parkinson disease; GVB: granulovacuolar degeneration body; LB: Lewy body; LBD: Lewy body disease; mitoQC: mitochondrial quality control; nbM: nucleus basalis of Meynert; PD: Parkinson disease; PDD: Parkinson disease with dementia; p-S65-Ub: PINK1-phosphorylated serine 65 ubiquitin; SN: substantia nigra; TLBD: transitional Lewy body disease; Ub: ubiquitin </p