Incorporation of molecular scissors to study dynamic conformation upon Parkin phosphorylation

A. Superimposition of WT-Parkin (PDB 5C1Z) and phospho-Parkin (PDB 6GLC) structures. RING2 (blue), pUbl (brown), RING0 (red), and ACT (black) are shown. For clarity, other Parkin domains are not included.

B. Schematic representation of Parkin domains and various constructs used in this study. HRV 3C and TEV sites incorporated in the Parkin construct are marked with black and green arrows, respectively.

C. Size-exclusion chromatography (SEC) assay shows the binding/displacement of Ubl-linker (1–140) under native or phosphorylated conditions. A colored key for each trace is provided. Coomassie-stained gels of indicated peaks are shown in the lower panel. A schematic representation is used to explain SEC data.

D. Size-exclusion chromatography (SEC) assay shows binding/displacement of RING2 (383–465) under native or phosphorylated conditions. Coomassie-stained gels of indicated peaks are shown in the lower panel. TEV as contamination is indicated (*).

Characterization of competing mode of binding between pUbl and RING2

A. SEC assay shows depletion of RING2 (383–465) from phospho-Parkin stabilize pUbl-linker (1–140) binding with Parkin (141–382) after treatment with 3C protease. Fractions that were pooled for subsequent proteolysis are highlighted in the box.

B. SEC assay shows depletion of pUbl-linker (1–140) from phospho-Parkin stabilize RING2 (383–465) binding with Parkin (R0RB, 141–382) after treatment with TEV protease. Fractions that were pooled for subsequent proteolysis are highlighted in the box.

C. Crystal structure of pUbl-linker (1–140) depleted Parkin (141–465) complex with pUb (brown). Different domains of Parkin are colored, as shown in the left panel. Catalytic C431 is highlighted. Structure of pUbl-linker (1–140) depleted Parkin (141–465)-pUb complex (colored as in the left panel) is superimposed with R0RBR structure (PDB 4I1H, grey) in the right panel. A schematic representation of the Parkin Q347C (3C, TEV) construct used for crystallization is shown at the bottom.

K211N mutation affects RING2 displacement, not pUbl

A. Size-exclusion chromatography (SEC) assay to test the displacement of RING2 (left panel) or pUbl-linker (right panel) after phosphorylation of Parkin K211N (3C, TEV).

B. Ubiquitination assay to test the activity of Parkin K211N in the presence of pUb or using phosphoParkin K211N. The middle panel shows a Coomassie-stained loading control. A non-specific, ATP-independent band is indicated (*). The lower panel shows Miro1 ubiquitination for the respective proteins in the upper lane. Coomassie-stained gel showing Miro1 is used as the loading control of substrate ubiquitination assay.

C. Crystal structure of pUbl-linker (1–140) depleted R0RBR (R163D/K211N)-pUb complex. The superimposed apo R0RBR structure (PDB 4I1H) is shown in grey. A schematic representation of the Parkin R163D/K211N/Q347C (3C) construct used for crystallization is shown at the top.

Untethering of the linker between IBR-RING2 allows Parkin and phospho-Ubl interaction in trans

A. Binding assay between phospho-Parkin K211N and AUbl-Parkin. A colored key for each trace is provided. Coomassie-stained gels of indicated peaks are shown in the lower panel. A schematic representation is used to explain SEC data. Isothermal Titration Calorimetry assay between phosphoParkin K211N and AUbl-Parkin is shown in the lower panel. N.D. stands for not determined.

B. Binding assay between phospho-Parkin K211N and untethered AUbl-Parkin (TEV). A colored key for each trace is provided. Coomassie-stained gels of indicated peaks are shown in the lower panel. A schematic representation is used to explain SEC data. Isothermal Titration Calorimetry assay between phospho-Parkin K211N and untethered ΔUbl-Parkin (TEV) is shown in the lower panel. The dissociation constant (Kd) is shown.

C. SEC assay to test binding between untethered R0RBR Q347C (TEV) and phospho-Parkin K211N (3C), and displacement of RING2 (383–465) from R0RBR, the left panel. The peak1 (black) containing R0RB (141–382) and phospho-Parkin K211N complex was incubated with pUb-3Br, followed by HRV 3C protease, to purify ternary trans-complex of phospho-Parkin (1–140 + 141–382 + pUb) on SEC, the right panel. The concentrated fractions from the shoulder (highlighted with a dashed line) of the peak in the right panel were loaded on SDS PAGE to confirm complex formation. A schematic representation of the Parkin constructs used for crystallization is shown at the bottom.

D. Crystal structure of the trans-complex of phospho-Parkin with pUb (brown) shows phospho-Ubl domain (wheat) bound to RING0 (cyan) domain of Parkin (cyan).

Parkin dimerization and trans-activation of native Parkin are mediated by phosphorylation of the Ubl domain of Parkin

A. SEC assay between phospho-Parkin and untethered AUbl-Parkin. A colored key for each trace is provided. Coomassie-stained gels of indicated peaks are shown in the lower panel. TEV protein contamination is indicated (*). A schematic representation of the Parkin constructs used for experiments in panels A and B is shown at the top.

B. Isothermal Titration Calorimetry assay between phospho-Parkin and untethered ΔUbl-Parkin (TEV). The dissociation constant (Kd) is shown.

C. SEC assay between phospho-Parkin and untethered WT-Parkin (TEV) (upper panel) or untethered Parkin K211N (TEV) (lower panel). A schematic representation of the Parkin constructs used for experiments in panels C and D is shown at the bottom.

D. SEC-MALS assay to confirm the complex formation between untethered WT-Parkin (TEV) and phospho-Parkin.

E. Ubiquitination assays to check the WT-Parkin activation (right panel) with increasing concentrations of phospho-Parkin T270R/C431A. A non-specific, ATP-independent band is indicated (*). The lower panel shows a Coomassie-stained loading control.

Analysis of Parkin mutant recruitment to mitochondria in HeLa cells

A. Immunofluorescence of HeLa cells co-transfected with either mCherry-Parkin wild-type (WT) or mCherry-Parkin S65A and GFP-Parkin C431F or, B. GFP-Parkin H302A/C431F and, C. GFP-Parkin K211N/C431F. Cells were treated for 1 hour with 10 μM CCCP, and DMSO was used as a control.Mitochondriawere labeled with anti-TOMM20 antibody (blue). Scale bar = 10 μm. D. Quantification of GFP-Parkin (WT and mutants) on mitochondria. The co-localization of GFP-Parkin (WT and mutants) with TOMM20 (mitochondria) was evaluated using Pearson’s correlation coefficient. Errors are represented as S.D. Statistical differences in Pearson’s correlation coefficient were evaluated using one-way ANOVA and Tukey’s multiple comparisons post-test. Statistical significance is as follows: *, p< 0.05; ****, p < 0.0001.

ACT plays a crucial role in enzyme kinetics

A. Size-exclusion chromatography (SEC) assay to test the binding of E2~Ubdon with phospho-Parkin (left panel) or phospho-Parkin AACT (right panel). Assays were done using Parkin in complex with pUb. A colored key for each trace is provided. Coomassie-stained gels of indicated peaks are shown in the lower panel. The upper panel shows a schematic representation of the Parkin ΔACT construct used.

B. Size-exclusion chromatography (SEC) assay to check displacement of the RING2 domain after phosphorylation of Parkin ΔACT. The upper panel shows a schematic representation of the Parkin ΔACT construct used for the RING2 displacement assay. Conformational changes in Parkin, as observed by the SEC experiment, are shown schematically.

C. Ubiquitination assay to check the effect of ACT deletion (ΔACT) on Parkin activity. A non-specific, ATP-independent band is indicated (*). The middle panel shows a Coomassie-stained loading control. In the lower panel, the bar graph shows the integrated intensities of ubiquitin levels from three independent experiments (mean ± s.e.m.). Statistical significance was determined using pair-wise student’s t-test (**P < 0.01, ***P < 0.001, ns-nonsignificant).

ACT is more efficient in cis

A. Crystal structure of ternary trans-complex of phospho-Parkin with pUb (1–140 + 141–382 + pUb), left panel. pUbl (wheat) and RING0 (cyan) of Parkin are shown. The right panel shows superimposed structures of ternary trans-complex of phospho-Parkin with pUb, colored as the left panel, and the phospho-Parkin complex with pUb (PDB 6GLC) is shown in grey.

B. SEC assay to check the binding between untethered ΔUbl-Parkin (TEV) and phospho-Ubl (1–76). A colored key for each trace is provided. Coomassie-stained gels of indicated peaks are shown in the lower panel.

C. Crystal structure of ternary trans-complex of phospho-Parkin with cis ACT (1–76 + 77–382 + pUb) shows ACT (cyan) present in the pocket on RING0 (Cyan) and pUbl (wheat) in the vicinity.

D. Comparison of R0RBR and ΔUbl-Parkin activation using the increasing concentrations of pUbl (1–76). A non-specific, ATP-independent band is indicated (*). The middle panel shows a Coomassie-stained loading control. The lower panel shows Miro1 ubiquitination for the respective proteins in the upper lane. Coomassie-stained gel showing Miro1 is used as the loading control of substrate ubiquitination assay.

E. Ubiquitination assay of ΔUbl-Parkin with increasing concentrations of pUbl (1–76), pUbl-linker (1–140), pUbl-linker-ΔACT (1–140, Δ101–109). A non-specific, ATP-independent band is indicated (*). The middle panel shows a Coomassie-stained loading control. The lower panel shows Miro1 ubiquitination for the respective proteins in the upper lane. Coomassie-stained gel showing Miro1 is used as the loading control of substrate ubiquitination assay.

F. Comparison of R0RBR and AUbl-Parkin activation using the increasing concentrations of pUbl (1- 76)/pUbl-linker (1–140)/pUbl-linker-ΔACT (1–140, Δ101–109). A non-specific, ATP-independent band is indicated (*). The middle panel shows a Coomassie-stained loading control. The lower panel shows Miro1 ubiquitination for the respective proteins in the upper lane. Coomassie-stained gel showing Miro1 is used as the loading control of substrate ubiquitination assay.

Linker (408–415) of Parkin binds with donor ubiquitin (Ubdon) of E2-Ubdon

A. The asymmetric unit of the crystal structure of pUbl-linker (1–140) depleted R0RBR (R163D/K211N)- pUb complex. Parkin molecule-1 (domains are shown in different colors) and pUb (brown) are shown. Parkin molecule-2 (grey) and pUb (orange) are shown. The interface of two Parkin molecules is highlighted (dashed line).

B. The 2Fo-Fc map (grey) of the linker region between REP and RING2. 2Fo-Fc map is contoured at 1.5 o. Water molecules are represented as w.

C. Crystal structure shows interactions between the linker (408–415) and ubiquitin. Different regions are colored as in panel A. Hydrogen bonds are indicated as dashed lines.

D. Sequence alignment of Parkin from various species highlighting conservation in the linker (408–415) region. Residue numbers shown on top of sequence alignment are according to human Parkin.

E. Ubiquitination assay of Parkin mutants in the linker region. The middle panel shows a Coomassiestained loading control. The lower panel shows Miro1 ubiquitination for the respective proteins in the upper lane. Coomassie-stained gel showing Miro1 is used as the loading control of substrate ubiquitination assay.

F. Size-exclusion chromatography (SEC) assay to compare the binding of E2~Ub with phospho-Parkin (upper panel) or phospho-Parkin I411A (lower panel). Assays were done using Parkin in complex with pUb. A colored key for each trace is provided. Coomassie-stained gels of indicated peaks are shown.

Model shows different modes of Parkin activation

The cis-activation model uses the binding of pUbl in the same molecule, thus resulting in the displacement of RING2 (1). The trans-activation model uses the binding of pUbl of fully-activated Parkin (phospho-Parkin complex with pUb) with partially-activated Parkin (WT-Parkin and pUb complex), thus resulting in the displacement of RING2 in trans (2). Recruitment and activation of Parkin isoforms lacking Ubl (Isoform 10) or RING2 domain (Isoform 5), thus complementing each other using the transactivation model (3). Catalytic cysteine on RING2 is highlighted.

Data collection and Refinement statistics

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