Structure of phosphorylated UBL domain and insights into PINK1-orchestrated activation

Jacob D. Aguirrea,1, Karen M. Dunkerleya,1, Pascal Merciera, and Gary S. Shawa,2

aDepartment of Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario, London, ON N6A 3K7, Canada

Edited by Angela M. Gronenborn, University of Pittsburgh School of Medicine, Pittsburgh, PA, and approved November 21, 2016 (received for review August 5, 2016) Mutations in PARK2 and PARK6 are responsible for the PINK1 stimulates parkin activity through phosphorylation of majority of hereditary Parkinson’s disease cases. These genes both Ub and parkin’s UBL domain at an equivalent serine 65 encode the E3 ubiquitin ligase parkin and the kinase PTEN- position in sequence and structure (16–21). Whereas each phos- induced kinase 1 (PINK1), respectively. Together, parkin and PINK1 phorylation event can increase parkin activity independently, regulate the pathway, which recycles damaged mito- maximal activity is obtained when both parkin and Ub are phos- chondria following oxidative stress. Native parkin is inactive and phorylated (18, 19). Phosphorylation of parkin at S65 increases exists in an autoinhibited state mediated by its ubiquitin-like parkin’s affinity for phosphoubiquitin (pUb) (14, 15, 22, 23). (UBL) domain. PINK1 phosphorylation of serine 65 in parkin’s UBL Three-dimensional structures show this optimizes a pUb binding and serine 65 of ubiquitin fully activate ubiquitin ligase activity; site, remote from the UBL site (15), and computational mod- however, a structural rationale for these observations is not clear. els predict local structural changes occur in the UBL domain Here, we report the structure of the phosphorylated UBL domain upon phosphorylation (24). Further, binding of pUb alloster- from parkin. We find that destabilization of the UBL results from ically induces a conformational change in parkin, liberating rearrangements to hydrophobic core packing that modify its struc- parkin from its autoinhibited state (14, 15, 22, 25). ture. Altered surface electrostatics from the phosphoserine group Although the functional effect of PINK1 on parkin activity is disrupt its intramolecular association, resulting in poorer autoin- well reported, the molecular interpretation of the UBL phos- hibition in phosphorylated parkin. Further, we show that phos- phorylation signal especially in conjunction with pUb is less phorylation of both the UBL domain and ubiquitin are required to clear. In this work, we present the solution structure of PINK1- activate parkin by releasing the UBL domain, forming an extended phosphorylated UBL (pUBL) from human parkin. We find that structure needed to facilitate E2–ubiquitin binding. Together, the altered thermodynamic stability and surface potential in pUBL results underscore the importance of parkin activation by the disrupt the autoinhibitory association in parkin. Further, we PINK1 phosphorylation signal and provide a structural picture of show phosphorylation of both UBL and Ub is needed to release the unraveling of parkin’s ubiquitin ligase potential. the UBL domain and form an extended structure where the E2- binding site is unprotected, allowing its recruitment during the E3 ligase | Parkinson’s disease | conformational change | ubiquitination cascade. phosphorylation | ubiquitin Significance osttranslational modifications are sophisticated biological P“switches,” relaying molecular signals to govern cellular pro- Parkinson’s disease is a devastating neurodegenerative disor- cesses and respond to external stimuli. Under conditions of der that can be inherited through mutations in genes encod- oxidative stress, two modifications in particular, phosphorylation ing the kinase PTEN-induced kinase 1 (PINK1) or the ubiqui- and ubiquitination, cooperate to regulate mitochondrial dynam- tin ligase parkin. Parkin exhibits neuroprotective properties ics and restore homeostasis (well reviewed in ref. 1). The ubiqui- by ubiquitinating on damaged mitochondria, leading tin (Ub) ligase parkin (encoded by the PARK2 ) and Ser/Thr to their turnover. However, parkin exists in an inactive state kinase PTEN-induced kinase 1 (PINK1, encoded by PARK6) that must be alleviated by PINK1 phosphorylation. Therefore, are important regulators of mitochondrial dynamics and muta- the molecular interpretation of the phosphorylation signal is tions in either gene are causative of familial forms of Parkin- immensely valuable to our understanding of parkin’s role in son’s disease (2, 3). Multiple studies show that in response mitochondrial maintenance and neuronal fidelity. We present to mitochondrial oxidative stress parkin activity is stimulated the 3D structure of the phosphorylated inhibitory domain of through phosphorylation by PINK1 (4, 5). This in turn facilitates parkin and describe the structural changes that lead to activa- parkin-mediated ubiquitination of several proteins at the outer tion of the enzyme. Alongside the available phosphoubiquitin mitochondrial membrane and signals the turnover of damaged structure, this study completes a structural picture of PINK1- mitochondria through the mitophagy pathway (6–8). Some muta- orchestrated parkin activation in impaired mitochondria. tions in parkin cause its dysfunction, leading to an accumulation Author contributions: J.D.A., K.M.D., and G.S.S. designed research; J.D.A., K.M.D., and of mitochondrial damage that appears to be especially detrimen- G.S.S. performed research; J.D.A., K.M.D., P.M., and G.S.S. analyzed data; and J.D.A., tal in neurons, a potential cause for Parkinson’s disease. K.M.D., and G.S.S. wrote the paper. Parkin belongs to the RBR subfamily of E3 ubiquitin ligases The authors declare no conflict of interest. (9) that function by transferring Ub from an E2-conjugating This article is a PNAS Direct Submission. enzyme to a substrate through an E3–Ub intermediate (10). Freely available online through the PNAS open access option. These enzymes are structurally autoinhibited in their native states Data deposition: The atomic coordinates and structure factors have been deposited in by unique accessory domains (11–13), indicating that RBR lig- the Protein Data Bank, www.pdb.org (PDB ID code 5TR5). The NMR chemical shifts and ases must be activated to carry out their full ubiquitination poten- restraints have been deposited in BioMagResBank, www.bmrb.wisc.edu (accession no. tial. Specifically, parkin contains an N-terminal ubiquitin-like 30197). (UBL) domain shown to inhibit Ub ligase activity (11). Three- 1J.D.A. and K.M.D. contributed equally to this work. dimensional structures of parkin show UBL associates with the 2To whom correspondence should be addressed. Email: [email protected]. C-terminal region (R0RBR) through both ionic and hydrophobic This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. interactions, blocking the proposed E2 recognition site (14, 15). 1073/pnas.1613040114/-/DCSupplemental.

298–303 | PNAS | January 10, 2017 | vol. 114 | no. 2 www.pnas.org/cgi/doi/10.1073/pnas.1613040114 Downloaded by guest on September 29, 2021 Downloaded by guest on September 29, 2021 gir tal. et Aguirre This core. protein the into by the buried confirmed whereas is solvent, was L61 Q64 into of and backbone sidechain Q63, the D62, aliphatic from of away atoms oriented sidechain are pUBL, in changes tural in F4 terminal with the bonds hydrogen temperature shortening 29). of increased (28, loss showed the bonding pSer65 reflecting of dependency, hydrogen amide amide the the of contrast, compared in In indicative Q64 site S4) and (Fig. phosphorylation Q63 ment S65 including the UBL, Amide with S3). near (Fig. decreases analysis large (∆ shift coefficients chemical temperature and struc- propensity, secondary constants, ture coupling backbone by is supported was reorganization new The 2 a (Fig. D62. L61–I66 ing hydroxyl residues, of upstream S65 amide the to the backbone translated between the bond and exposes hydrogen group and a rotated eliminates conformational is This change phosphory- (pSer65). backbone 65 S65 phosphoserine protein of the sidechain the S2 the near where (Fig. occur site structures changes lation crystal structural UBL obvious mouse B), and human the and I23–Q34; (α1, α-helices R6; (26) PINK1 of orthologue S65, at insect phosphorylated active fully ( catalytically domain a UBL using parkin human Stability. the Its Decreases UBL Parkin of Phosphorylation Discussion and Results ouinsrcueo UL sn M pcrsoy(i.2A (Fig. cal spectroscopy the NMR determined using we and pUBL, parkin, of with this structure how association solution establish UBL and the phosphorylation modifies of UBL. effects Parkin structural Phosphorylated the of Structure NMR Solution per- stability. that thermodynamic and changes pathway structural folding in its that turb results show 1 UBL results (Fig. These of altered. phosphorylation UBL is pUBL with of surface compared hydrophobic pUBL pres- of the Interest- in ence fluorescence phosphorylation. greater markedly upon showed bis-ANS hydrophobicity ingly, in 4,4’-bis-1- changes probe used for to we fluorescence UBL, and stable (bis-ANS) than less anilinonapthalene-8-sulfonate aggregation is pUBL to Because susceptible unfold- 1B). earlier more (Fig. much urea a in undergoing transition UBL, ing than stable signif- less was icantly pUBL 1A confirmed (Fig. experiments denaturation UBL chemical than the lower temperature of a showed characteristic transition pUBL 1A), treated thermally (Fig. However, nm fold. 222 ubiquitin observed and forms and minima nm UBL with disease 205 for signatures near several spectra spectral (CD) for similar dichroism showed reported Circular pUBL been (27). UBL of has hypothe- the stability as of We the fold, precipitation. impacted UBL to negatively the phosphorylation prone that more sized was and solubility i.S1 Fig. A MRE (deg cm2 dmol -1 ) β -10000 10000 .Or2 oeteeg tutrsfr canoni- a form structures lowest-energy 25 Our S1). Table β gapuiutnlk od four-strand a fold: ubiquitin-like -grasp ,F13–V17; 2, hra eauain Bis-ANSfluorescence Thermal denaturation 0 A -,L1Q4 that L61–Q64) (α2-L, element structural α-helix–like 0 1 2 3 4 250 240 230 220 210 200 and wavelength (nm) .Cmae ihUL ULhddecreased had pUBL UBL, with Compared B). 1 β H- ,L1I4 and L41–I44; 3, 15 eeoula O measurements NOE heteronuclear N β tad sarsl fteestruc- these of result a As strand. 4 ,V6C9.I oprsnwith comparison In V56–C59). α2, δ NH B

Fraction unfolded /∆ 0.0 0.5 1.0 Urea denaturation )frpB loshowed also pUBL for T) 0246 3.30 ±0.02M β ,V7Q1 n two and V67–Q71) 4, M Urea - tutrlele- structural α2-L and ,sgetn the suggesting C), β 4.43 ±0.02M set(β -sheet and S1C) Fig. eproduced We ,form- A–C), oidentify To β ,M1– 1, C A -grasp and

Bis-ANS fluorescence (AU) β 1000 2000 1, 0 5 0 5 600 550 500 450 niaeta h oiainntoki ULi lee pnS65 upon altered is experiments pUBL in These network ionization 31). the that (30, amide indicate phosphoproteins pSer65 for the pK of observations similar titration a the calculating measuring proton, the by from this phosphate confirmed pSer65 ionization the reporting be of might this suspected in we results structures, pUBL data titration L61 also pK the is a of environment Fitting UBL) chemical ionization. not by their (but affected suggesting pUBL in pH, L61 with of titrated groups methyl 2F sidechain (Fig. H68, 5.97 pK to to the unit 0.5 on by effect increased no has titrations phorylation pH H performed struc- pK the tracking we calculated the this, all measure in investigate of to pSer65 further tautomer toward To N2-protonated oriented tures. chem- the N2 idea, with confirmed this H68, with analysis Consistent the shift H68. and or pSer65 ical R6 on of group 2B sidechains may phosphate interaction basic (Fig. negative ionic the H68 transient between a and that exist R6, hypothesized We I66, S6). between Fig. observed the 4.8 NOEs of distance: most tiple in (average H68 structures pUBL. and R6 calculated in toward directed Network is phosphate Ionization pSer65 the Modifies functional pSer65 and physical in pUBL. and differences pUb between key the properties backbone an of the provide orientation the for observations the explanation These to maintaining residues. pSer65 preserved, backbone is surrounding from Q62 bond pUb nearly of In amide is hydrogen (21). pUb S2C) structural (Fig. of state the structure unphosphorylated the overall to the identical where structure, upon crystal pUBL are in I66 hydrophobicity of modified atoms phosphorylation. showed exper- methyl bis-ANS that sidechain the reaffirm iments the chem- changes structural large to These addition, observed. changes In from upon structure. space shift L61 our in ical and in distant group L50, being phosphate A46, despite the I23, 2D) V3, (Fig. M1, phosphorylation contrast, of In positions S7 ). resonance (Fig. core hydrophobic report the not chemi- 13 to do these change large (22), this phosphorylation several upon report changes shift data cal parkin (HSQC) to in coherence region suggested Whereas quantum linker region (24). the the with activation widening to during por- cleft close a for core uncovers important hydrophobic solvent be bulk this sidechain the of and into 2E tion pSer65 (Fig. backbone of exists residues movement extensive L61 aliphatic The by around these centered supported between and NOEs as V67 pUBL and V56, in L50, A46, F45, from I66 and pSer65 releases D62–Q64 and the loop in flexibility increases phosphorylation showed that wavelength (nm) SCsetadmntaeetniecagsi h methyl the in changes extensive demonstrate spectra HSQC C h tutr fpB tnsi tr otatt h pUb the to contrast stark in stands pUBL of structure The I23, V5, V3, M1, residues comprising core hydrophobic A a f64 Fg 2F (Fig. 6.41 of p UBL UBL inl in signals PNAS a ae rae xoe yrpoi ufc than surface UBL. hydrophobic for exposed greater indi- a pUBL for cates fluorescence increased An pUBL. and (C midpoint ration at measured as 5 denaturation urea by pUBL and at UBL collected ther- line), and (dashed 5 line) states (solid denatured native mally showing and (black) UBL (red), UBL of the pUBL spectra CD destabilizes (A) parkin. phosphorylation of domain S65 1. FIG. fH1adH8i B n ULby pUBL and UBL in H68 and H11 of .BcueL1i itn rmH8i our in H68 from distant is L61 Because ). ◦ ◦ i-N ursec ntepeec fUBL of presence the in fluorescence Bis-ANS ) yC litct t22n.Osre denatu- Observed nm. 222 at ellipticity CD by C 75 and C | 1 H- aur 0 2017 10, January 13 ◦ SCseta hra phos- Whereas spectra. HSQC C nodn rfieof profile Unfolding (B) respectively. C, a 1 au f64,cnitn with consistent 6.47, of value ± H- to −1 .W oe hti addition in that noted We ). Ei niae eieec curve. each beside indicated is SE a 15 β fH1 h pK the H11, of eeoula single- heteronuclear N ( 4 | hresae We state. charge −2 i.S5 Fig. o.114 vol. ) wn omul- to owing A), ˚ npB,the pUBL, In ). and | o 2 no. a i.S6). Fig. fH68 of | 1 and 299 H-

BIOCHEMISTRY A BC

D E

F

Fig. 2. Solution structure of pUBL. (A) Structure of Ser65-phosphorylated pUBL determined by NMR spectroscopy. Structure closest to average is represented as a cartoon with secondary structure as described in the text. (B) Expanded view of pUBL structure around pSer65. (C) UBL structure (extracted from PDB ID 5C1Z) is shown for comparison. (D) Chemical shift perturbations in methyl (CH3) resonances upon Ser65 phosphorylation. Resonance assignments for methyl groups in pUBL are indicated (red spectrum), reporting changes to the hydrophobic core upon phosphorylation. (E) Expanded view of pUBL structure showing hydrophobic residues affected by phosphorylation. (F) Chemical shifts observed during the pH titration of UBL (black curves) and pUBL (red curves). 1 H chemical shifts were measured at each pH and fitted to a modified Henderson–Hasselbalch equation by nonlinear regression to determine pKa ± SE (indicated beside the curve). Titration of S65 and L61 in UBL was not observed.

phosphorylation and provide evidence that pSer65 partially neu- However, pSer65 is brought into close proximity of the RING1 tralizes the H68 positive charge. domain, specifically the carboxylate group of D274, resulting in electrostatic repulsion and disruption of the polar interactions Phosphorylation Alters the UBL Association in Parkin. Given that of D274 with R6 and H68 (Fig. 3C). These changes to electro- parkin autoinhibition is maintained through intramolecular inter- static interactions in the binding pocket provide a rationale for actions with the β-sheet face and I44-patch of UBL, we sought to the poorer autoinhibition observed in phosphorylated parkin. examine how phosphorylation might directly perturb this bind- To further examine how phosphorylation of the UBL domain ing interface. We previously observed that S65 phosphoryla- modulates its interaction with parkin, we probed the pUBL– tion results in ∼10-fold weaker binding of pUBL vs. UBL when R0RBR interaction by transverse relaxation optimized spec- measured in trans (26 µM vs. 3.8 µM Kd) (15). To investigate troscopy (TROSY) from the perspective of R0RBR. Consistent the structural basis of the weakened affinity, we calculated the with the calorimetric data, NMR experiments showed pUBL is surface electrostatic potential of the UBL–R0RBR binding inter- still able to bind parkin in trans, despite the weakened affinity. face (32). On its parkin-interacting surface, UBL has an over- Many of the perturbed residues in RING1 and IBR are affected all positive surface that interacts with an overall negative surface in a similar fashion for binding of pUBL and UBL, indicating on R0RBR (Fig. 3A, Left). Upon phosphorylation, the predom- that the RING1/IBR interface is still used for pUBL recruit- inantly −2 charge of the O-phosphate group changes the sur- ment (Fig. 3D and Fig. S8). Further, we did not observe chem- face to a more negative state by partially neutralizing charges on ical shift changes in RING0 or RING2 upon binding of pUBL, the β-sheet face of pUBL (Fig. 3A, Right). The additional neg- indicating the arrangement of these domains is not modified nor ative potential on the pUBL surface is unfavorably brought into does pUBL interact specifically with either domain (Fig. S8). The close proximity to the negative binding pocket on R0RBR. We most significant changes in chemical shift upon phosphorylation generated a model of pUBL-bound parkin, maintaining the I44- occur in the tether region (residues F381–T415) connecting the centered binding interface observed in crystal structures (14, 15) IBR and RING2 domains (Fig. 3D). Much of this region is flex- (Fig. 3 B and C). In this model, I44 and V70 maintain contact with ible based on heteronuclear NOE experiments (15) and is unre- L266, as their aliphatic sidechains show minimal change in struc- solved in crystal structures (15, 35). Chemical shift perturbations ture and chemical environment upon phosphorylation (Fig. 2D). show that a portion of this tether (residues F381–E385) lies near

300 | www.pnas.org/cgi/doi/10.1073/pnas.1613040114 Aguirre et al. Downloaded by guest on September 29, 2021 Downloaded by guest on September 29, 2021 rpsdpB idn nefc.Rsdenmeig r ooe codn oterrsetv oan.pe6 sbogtit ls rxmt fD274 of proximity close into brought is pUBL pSer65 with domains. 142–465) respective residues 5C1Z, their ID to (C (PDB according Chimera. UCSF R0RBR colored in parkin are Modeler of numberings using modeled architecture Residue (<5 were domain interface. (A383–A390) Overall residues binding tether (B) (Left pUBL Unresolved PyMOL. UBL proposed region. in R0RBR, binding UBL visualized (positive). the blue and in and (32), aligned (neutral), APBS white and (negative), PDB2PQR red programs in color by indicated 3. Fig. gir tal. et Aguirre in parkin might full-length phosphorylation of how structure examine the To alter parkin. phosphory- of in S65 constructs conducted of been impact Structure. have the Parkin lation evaluate Extended to an experiments to physical Leads Activation pUb to (15) remodeling for site conjugate. tether E2–Ub pUb-binding the incoming of the the region accommodate optimizes upstream the It primes phosphorylation purpose: and UBL dual site that binding conceivable a E2 is pro- serves the it is blocking Therefore, and by 36). W403 activity (35, includes ligase shift suppress that chemical to region observe posed a not R396, do this beyond we near Notably, changes likely tether. is the pSer65 of reporting that portion indicate specifically and are moiety these phosphate 3D the suggest (Fig. G385–E395, parkin in residues R0RBR in residues into several titrated for is and pUBL shifts when mag- chemical the tether of in the changes direction pronounced and more phospho- to nitude However, leads state. UBL autoinhibited of spectra the rylation beneath in shown domain are UBL (G375–T415) the residues tether of resonances amide CSP as for calculated plots were (CSP) CSPs perturbation experiment. respective shift the Chemical for 15. colored ref. and in affinities to according (purple, UBL in eetd600-MHz Selected (D) repulsion. charge of source possible a A), ˚ .Teedfeecswe ULi on,especially bound, is pUBL when differences These S8). Fig. C B A ufc hreis charge Surface (A) R0RBR. with interactions autoinhibitory disrupt UBL of phosphorylation upon potential electrostatic surface to Changes IBR IBR A383 Left RING1 pSer65 n UL(red, pUBL and ) T387 R6 D274 H68 RING1 pUBL V70 Right sn ULadR0RBR and pUBL using trans, B pUBL UBL T270 neatoswt h ehrdmi.ULadpB neatoswere interactions pUBL and UBL domain. tether the with interactions ) I44 A390 IBR L266 pSer65 R42 Tether cis RING1 D262 n oidentify to and D394 otbio- Most RING0 1 RING2 H- [∆δ = 15 W403 RS SCsetaof spectra HSQC TROSY N HN 2 etto aems eofe yacag nhydrodynamic in (f/f sedi- change coefficient the a frictional in by the increase in offset modest reflected be the shape, must the kDa), to rate (∼9 due mentation expected pUb Whereas is blue). of coefficient 4A, addition sedimentation (Fig. sediment in S 4A). increase to 4.02 (Fig. an at found pParkin ultracentrifugation reproducibly and by was faster parkin pParkin/pUb for contrast, data In experimental the R with S, Results (3.86 34). approach 21, this (15, from structures 3D autoin- available of using properties parkin, hydrodynamic hibited calculated we studies, 4 with these (Fig. associated con- domains is are the domain R0RBR parkin (pUBL) under the UBL shape of the compact where forms tested same ditions both the retain indicating and respectively), monomeric S, (S 3.88 rates and and profiles pParkin sedimentation and similar parkin by displayed Both 23). complexes (15, chromatography pParkin/pUb exclusion size homogenous S65-phosphorylated and fully (pParkin), native, purified parkin of analytical We shape solution. used hydrodynamic in we the parkin probe this, to modulates (AUC) further ultracentrifugation activation pUb how + D 0.2∆δ CSP (ppm) 15 N (ppm) 15 N (ppm) 15 N (ppm) 115.0 115.0 116.0 116.0 124.0 123.0 122.0 124.0 126.0 125.0 0.00 0.05 0.10 0.15 0.20 N 2 ,adpB (Right pUBL and ), ] 0. R0RBR +UBL R0RBR V423 R455 8.4 F381 V278 8 9 0 410 400 390 380 5 ulCPposaefudin found are plots CSP Full . D346 8.4 W403 C212 9.0 8.8 F384 Residue 1 1 1 H (ppm) H (ppm) H (ppm) 2 A390 T387 H, A383 PNAS 15 -aee aknRRRhglgtn differences highlighting R0RBR parkin N-labeled T270 F208 8.2 Q282 K412 8.2 | eecluae eaaey sn h online the using separately, calculated were ) V244 I298 T388 aur 0 2017 10, January 6 and ∼96% lc n e) ocomplement To red). and black A, G

= 15 15 15 CSP (ppm) N (ppm) N (ppm) N (ppm) 124.0 123.0 122.0 126.0 124.0 125.0 115.0 115.0 116.0 116.0 0.00 0.05 0.10 0.15 0.20 27 i.S8. Fig. )mthdnal perfectly nearly matched A) ˚ R0RBR +pUBL R0RBR 8.4 xaddve of view Expanded ) V423 V278 F381 8 9 0 410 400 390 380 2 auae,respectively, saturated, ∼92% W403 8.4 D346 C212 | 9.0 8.8 F384 1 Residue o.114 vol. 1 1 H (ppm) H (ppm) H (ppm) A390 A383 T387 0 T270 .Ide,f/f Indeed, ). V244 F208 K412 8.2 | Q282 I298 20,w 8.2 T388 o 2 no. showing B, = .6S 3.86 | 301 0

BIOCHEMISTRY A B

3.6 S 1.0 S 20,w f/f0 MW (Da) Parkin 3.86 1.31 52,163 pParkin 3.88 1.30 52,243 pParkin/pUb 4.02 1.37 61,143 0.5 3.5 S 3.8 S Normalized c(s) normalized c(s) normalized

3.4 S 4.2 S 0.0 4.3 S 0.0 0.5 2.5 3.0 3.5 4.0 4.5 SedimentationSedimentation Coefficient (S) (S) C 25 Most Most 3.7 S 20 extended compact pParkin/pUb

15

10 4.1 S

% of models % of 5 3.9 S % of models 0 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.0 S S (S) S 20,w (S)

Fig. 4. Hydrodynamic shape analysis of PINK1-activated parkin. (A) Sedimentation velocity experiments show little change in hydrodynamic shape upon UBL phosphorylation. A more extended conformation is observed only in the presence of pUb (higher f/f0). Example surface representations for the given hydrodynamic shapes are shown. (B) Ten representative pParkin/pUb models with a “displaced” pUBL domain. Two hundred models were generated using the ensemble optimization method (33), allowing pUBL linker to sample random conformations relative to R0RBR/pUb complex. R0RBR/pUb complex is represented as dark/light gray according to PDB ID 5CAW. Modeled pUBL-linker structures are shown according to their sedimentation coefficients as in C. (C) Distribution of sedimentation coefficients of 200 generated models of activated pParkin/pUb and autoinhibited pParkin/pUb. S20,w values were determined by HYDROPRO, using a residue-level shell model (34). Histogram x axis shows calculated S20,w values grouped by ±0.05 S.

increased from 1.30 to 1.37 with the addition of pUb, indicating ible linker (residues K76–R140), the tether region (E382–T414), pParkin/pUb adopts a more extended conformation in solu- and multiple loops in the protein (Fig. S9A). Remarkably, the tion. Hydrodynamic calculations using the autoinhibited struc- addition of pUb to pParkin resulted in the complete appearance ture (15) with pUb bound to the observed pUb binding site (22) of amide resonances assigned to pUBL (Fig. S9B) due to a much yielded a sedimentation coefficient of 4.56 S, much larger than longer T2 of these atoms after displacement from R0RBR. This observed experimentally (4.02 S). Together these results show result supports our sedimentation velocity experiments that show that pParkin/pUb does not maintain a compact structure. the full extrusion of the UBL domain occurs only upon PINK1 To determine the position of pUBL in the pParkin/pUb phosphorylation and recruitment of pUb. complex, we generated 200 models where pUBL is ran- domly displaced from R0RBR by its disordered 65-residue Conclusion linker (residues G77–I142) (33). Visual inspection shows that Autoinhibition of parkin activity by its UBL domain is a fas- pUBL samples the available conformational space surround- cinating evolutionary result to suppress substrate binding in a ing R0RBR in different models (Fig. 4B) and occupies a ubiquitin ligase enzyme. Parkin’s UBL shares high sequence and Gaussian distribution of sedimentation coefficients that aver- structural conservation with Ub, yet is significantly less ther- ages 3.89 S (Fig. 4C). The most extended model had a sedi- modynamically stable (27). We show PINK1 phosphorylation mentation coefficient of 3.36 S, reflecting pUBL displaced by further destabilizes the UBL by modifying its secondary struc- ˚ a near-linear linker (RG = 54 A). In contrast, the most com- ture, hydrogen bonding, and hydrophobicity, creating a new pact model observed was autoinhibited pParkin/pUb (4.56 S, secondary structural element (α2-L) near the phosphorylation RG = 27 A).˚ These observations are consistent with molecular site proposed to be important for interactions with the linker dynamics calculations for compact and extended forms of parkin based on computational studies (24, 37). The significant struc- (24). Taking all calculated structures into account, the observed tural differences in pUBL compared with pUb show why it can- sedimentation coefficient (4.02 S) for pParkin/pUb is most con- not compete for the pUb-binding site, yet can independently sistent with an average structure whereby pUBL is displaced increase parkin activity, if only partially. We show the orien- from R0RBR with RG ≈ 32 A,˚ occupying a range of positions tation of the phosphate group toward H68 and the R0RBR dictated by the configuration of the disordered linker. binding interface alters the autoinhibitory interaction and pro- To support our model that phosphorylation of parkin and vides a rationale for the weakened affinity observed in trans engagement by pUb are needed to release the pUBL domain, (14, 15, 22). Further, our results add to the growing consensus 1 15 we collected a series of T2-filtered H- N HSQC experiments that it is primarily the pUb signal that is responsible for the con- 15 for parkin, pParkin, and pParkin/pUb. Because backbone N formational change that relieves UBL-mediated autoinhibition atoms in parkin have a short bulk T2 (20 ms) consistent with (14, 15, 18, 22, 38). Although the specific order of phosphory- its 52-kDa size, we used a short T2 relaxation period to effec- lation events in cells remains controversial, parkin has evolved tively suppress most signals arising from the structured domains such that each phosphorylation signal induces a positive effect on of parkin. As expected, the resulting 1H-15N HSQC spectrum of activity and the receptors for each signal are physically distinct. parkin showed only a series of sharp signals arising from the flex- Therefore, the proposed mechanism of activation is consistent

302 | www.pnas.org/cgi/doi/10.1073/pnas.1613040114 Aguirre et al. Downloaded by guest on September 29, 2021 Downloaded by guest on September 29, 2021 0 ezlD,LsonvA roi S lvtR 21)UC7ratvt rfiereveals profile reactivity UBCH7 (2011) RE Klevit PS, Brzovic A, Lissounov DM, Wenzel 10. 1 huueV,e l 21)Atrglto fPri ciiytruhisubiquitin-like its through activity Parkin of Autoregulation (2011) al. et VK, Chaugule 11. 6 odpliC ta.(02 IK satvtdb iohnra ebaepotential membrane ligase mitochondrial by E3 activated the is primes PINK1 (2012) state al. autoinhibited et the C, Parkin. of Kondapalli of Disruption 16. activation (2015) the al. in et switch A, ubl/ubiquitin Kumar A 15. (2015) al. et V, Sauve 14. Autoin- ligase: ubiquitin RING-IBR-RING a HHARI, of Structure (2013) al. et DM, (2012) Duda K 13. Rittinger E, Christodoulou MG, Koliopoulos AC, Morris-Davies B, Stieglitz 12. 7 hb-uuhm ,e l 21)PN1mdae hshrlto fteParkin the of phosphorylation PINK1-mediated (2012) al. et K, Shiba-Fukushima 17. 9 alukieA ta.(04 akni ciae yPN1dpnetphosphorylation PINK1-dependent by activated is Parkin (2014) al. et A, Kazlauskaite Parkin. activate 19. to PINK1 by phosphorylated is Ubiquitin (2014) al. et F, Koyano 18. 0 aeL,e l 21)PN1popoyae bqii oatvt aknE ubiquitin E3 Parkin activate to ubiquitin phosphorylates PINK1 (2014) al. et LA, Kane 20. 2 ae ,SmckM cuetA oadrD(05 ehns fphospho- of Mechanism (2015) D Komander A, Schubert M, structure, Simicek ubiquitin T, affects phosphorylation Wauer Ser65 22. Ubiquitin (2015) al. et T, Wauer 21. 5 aaoK ta.(05 ieseicitrcinmpigo hshrltdubiquitin phosphorylated of mapping interaction Site-specific and (2015) al. domain et K, UBL Yamano the 25. releases PINK1 by Phosphorylation (2014) al. mecha- et feedforward TR, a Caulfield reveal 24. proteomics Quantitative (2014) al. et A, Ordureau 23. 6 odofH,e l 21)Dsoeyo aayial cieotoouso h Parkin- the of orthologues active catalytically of Discovery (2011) al. et HI, Woodroof 26. 7 aaiS,Sa S(07 ies tt uainuflstepri ubiquitin-like parkin the unfolds mutation state disease A (2007) GS Shaw SS, Safadi 27. adpooosadwtrrfie nXPO-I 4) lcrsai calcula- Electrostatic stan- (40). X-PLOR-NIH using in (39), refined CYANA water in determined and assignments was protocols NOE pUBL dard automatic on of collected and Structure were manual spectrometer. data using NMR 600-MHz 15. Inova ref. Varian in a described as in purified provided and expressed are methods Full Methods and Materials phosphorylation-activated of complex parkin’s model of function. understanding the unique a provide and release struc- UBL pUBL here The (23). solved ubiquitina- cascade mitophagy parkin ture the induce in stage rapidly any at to tion model feed-forward a with gir tal. et Aguirre .Srt E adnH hwG 21)RRE bqii iae:Nwsrcue,new structures, New ligases: ubiquitin E3 RBR (2014) GS Shaw H, Walden DE, Spratt response in ubiquitylome 9. PARKIN-dependent the of Landscape (2013) al. et SA, Sarraf 8. mito- PINK1-PARKIN The (2015) JW Harper J, Rinehart JA, to Paulo receptors A, Ordureau recruits JM, Heo PINK1 kinase 7. ubiquitin The (2015) al. et M, recruits Lazarou depolarization 6. mitochondrial by selectively stabilized PINK1 recruited (2010) is al. Parkin et N, (2008) Matsuda RJ 5. Youle DF, Suen A, Tanaka D, juvenile Narendra recessive autosomal cause 4. gene parkin the in Mutations (1998) muta- al. by et T, caused Kitada disease Parkinson’s 3. early-onset Hereditary (2004) in al. fidelity et mitochondrial EM, and Valente parkin, PINK1, 2. of roles The (2015) RJ Youle AM, Pickrell 1. aknadHAIt eRN/EThybrids. RING/HECT be to HHARI and parkin questions. new insights, depolarization. mitochondrial to and mitophagy. recruitment promote OPTN/NDP52 to of activation program TBK1 a drives pathway ubiquitylation chondrial mitophagy. induce mitophagy. for Parkin latent activates and Biol mitochondria damaged to Parkin autophagy. their promotes and 803. mitochondria impaired to parkinsonism. PINK1. in tions disease. Parkinson’s domain. eoaiainadsiuae aknE iaeatvt ypopoyaigSrn 65. Serine phosphorylating by activity Biol ligase Open E3 Parkin stimulates and depolarization catalysis. and activation for parkin mechanism. 34(20):2492–2505. ligation into insights and E3 Ariadne-family 21(6):1030–1041. an of hibition intermediate. thioester a via chains 13(9):840–846. ubiquitin linear synthesizes LUBAC bqii-iedmi rmsmtcodiltasoaino aknadregulates and Parkin of translocation mitochondrial mitophagy. primes domain ubiquitin-like fuiutna Ser65. at ubiquitin of 510(7503):162–166. iaeactivity. ligase bqii-nue AKNactivation. PARKIN ubiquitin-induced hydrolysis. and assembly chain oucvrPri activation. Parkin uncover to Parkin. ligase ubiquitin E3 Biol the of opening conformational the initializes synthesis. chain ubiquitin and 56(3):360–375. translocation PARKIN mitochondrial for nism o’ ies iaePN1 nlsso usrt pcfiiyadipc fmutations. of impact and specificity substrate Biol of Open Analysis PINK1: kinase disease son’s domain. 189(2):211–221. 10(11):e1003935. MOJ EMBO Biochemistry 2(5):120080. 1(3):110012. c Rep Sci Nature elBiol Cell J Science 30(14):2853–2867. Nature 2:1002. Neuron ice J Biochem 392(6676):605–608. 46(49):14162–14169. 304(5674):1158–1160. ice J Biochem 205(2):143–153. 524(7565):309–314. 85(2):257–273. ilChem Biol J MOJ EMBO 460(1):127–139. Nature rtiswere Proteins Methods. and Materials SI MOJ EMBO 458(3):421–437. Nature 34(3):307–325. o Cell Mol 496(7445):372–376. 290(42):25199–25211. 34(20):2506–2521. Nature 524(7565):370–374. 60(1):7–20. 474(7349):105–109. elBiol Cell J LSComput PLoS 183(5):795– MORep EMBO Structure o Cell Mol MOJ EMBO Nature Cell J 5 oo ,e l 20)Cytlsrcueadmlclrdnmc iuainof simulation dynamics molecular and structure Crystal (2008) al. et NMR protein K, of simplification Tomoo The (1975) 55. P Wright R, Williams C, Dobson I, Campbell phosphopeptide-complexed and 54. free a of dynamics Backbone (1994) al. et NA, Farrow Ha-Hb of 53. measurement for narrowing line Multiple-quantum (1995) A Bax S, Grzesiek measuring for 52. approach new A correlation: J Quantitative (1993) A Bax GW, Vuister biomolecular for 51. indicators pH Internal states. (2008) BD liquid Sykes and TC, Williams gas OK, in Baryshnikova water 50. of shift resonance Proton (1966) the JC for Hindman method simple 49. A index: chemical-shift 13C The (1994) BD structure Sykes DS, secondary Wishart of Sensitivity 48. (2006) JD Forman-Kay Z, Jia VK, Singh JA, predicting for Marsh method hybrid 47. A TALOS+: (2009) A Bax G, Cornilescu F, Delaglio Y, Shen 46. 2D active- the by of states proteins Tautomeric (1993) in S Roseman tautomers ND, Meadow histidine DA, Torchia JG, of Pelton Identification 45. (2003) al. et and JL, visualization the Sudmeier for program system 44. computer A processing View: NMR spectral (1994) R multidimensional Blevins B, A Johnson NMRPipe: 43. (1995) al. et F, Delaglio veloc- sedimentation 42. by molecular macromolecules NMR of analysis for Size-distribution Xplor-NIH (2000) P Using Schuck (2006) 41. GM Clore JJ, Kuszewski struc- CD, NMR Schwieters for of dynamics 40. dissection angle Torsion systematic (1997) K enable Wuthrich ligases C, E3 Mumenthaler P, ubiquitin Guntert of 39. Probes Parkin. ligase (2016) ubiquitin al. E3 et the KC, of Pao Activation 38. (2015) ligase W ubiquitin Springer F, for Fiesel mechanisms T, reveals reveals Caulfield ligase Parkin 37. of ubiquitin Structure E3 (2013) Parkin al. of et function JF, Trempe and Structure 36. (2013) other al. and et hydrodynamic BE, of Riley Prediction 35. (2011) JG Torre la de D, Amoros of A, modelling Ortega ensemble Advanced 34. (2015) DI Svergun M, Kachala HDT, Mertens G, Tria 33. nanosys- of Electrostatics (2001) JA McCammon MJ, Holst S, Joseph D, Sept NA, calcium- Baker extracellular amino 32. phosphorylated An of shifts chemical phosphoprotein: Random-coil (1999) Dentin KJ Lumb (1977) EA, Bienkiewicz T 31. Glonek A, Veis S, Lee gradients temperature 30. the from information Extracting (1997) in al. shifts et chemical NH, 1H Andersen of dependence 29. Temperature (1997) MP Williamson NJ, Baxter 28. his(....JDA hnsPrisnCnd o support. for Canada Parkinson thanks Research J.D.A. optimizing Canada (G.S.S.). and Chairs G.S.S.) for of (to Institutes MOP-14606 Rusal We Canadian Grant operating a Michele manuscript. Research by Health and funded the was expertise work of This AUC protocols. reading phosphorylation for careful Briere for Lee-Ann Barber thank Kathryn and Chaugule, ACKNOWLEDGMENTS. in calculated were coefficients sedimentation unstructured and with (34). method gyration HYDROPRO optimization pParkin/pUb of ensemble of Radii the using Models (33). generated (41). were experiments 77–142 Sedfit AUC residues 15. (p)UBL in ref. of in analyzed described experiments as were Interaction performed were (32). parkin APBS R0RBR and using performed were tions bqii-iedmi fmrn Parkin. murine of domain ubiquitin-like spectra. relaxation. NMR 15N by studies domain 2 homology Src proteins. enriched isotopically in couplings J proteins. 15N-enriched Soc in Chem constants coupling J(HNHa) three-bond homonuclear NMR. Phys data. chemical-shift 13C using NMR structure secondary protein of identification Implica- gamma-synuclein: fibrillation. and for alpha- tions between differences sequence to propensities shifts. chemical NMR from angles torsion backbone Sci Protein III unphosphorylated from and protein phosphorylated of histidines site NMR. correlated one-bond 1H/13C(delta2) data. NMR of analysis pipes. UNIX on based modeling. equation lamm and ultracentrifugation ity determination. structure DYANA. program new the with calculation ture activation. parkin Trans Soc Biochem activation. ligases. HECT and RING of aspects models. residue-level and atomic- from J proteins rigid of properties solution scattering. solution X-ray using macromolecules flexible ribosome. the and microtubules 98(18):10037–10041. to Application tems: acids. protein. binding averaging. conformational of importance Soc The Chem 1. Am shifts. J chemical NH polypeptide of proteins. 101(4):892–898. 44(12):4582–4592. 4(2):171–180. imlNMR Biomol J imlNMR Biomol J ESLett FEBS imlNMR Biomol J 115(17):7772–7777. Science 2(4):543–558. sn w-iesoa eeoula M techniques. NMR heteronuclear two-dimensional using coli, Escherichia 119(36):8547–8561. Biochemistry a hmBiol Chem Nat 57(1):96–99. 340(6139):1451–1455. 43:269–274. 41(1):5–7. 15(3):203–206. rti Sci Protein imlNMR Biomol J imlNMR Biomol J PNAS 9(4):359–369. etakHlnWle,Au ua,Viduth Kumar, Atul Walden, Helen thank We rgNc anRsnSpectrosc Reson Magn Nucl Prog 16:2971–2979. | 15(12):2795–2804. 12(5):324–331. aur 0 2017 10, January a Commun Nat 6(3):277–293. 4(5):603–614. ici ipy Acta Biophys Biochim mCe Soc Chem Am J mCe Soc Chem Am J 4:1982. o Biol Mol J imlNMR Biomol J Biochemistry ipy J Biophys | IUCrJ o.114 vol. rcNt cdSiUSA Sci Acad Natl Proc 48:47–62. 125(28):8430–8431. 273(1):283–298. Glc 117(19):5312–5315. 2:207–217. 1784(7-8):1059–1067. signal-transducing a , 78(3):1606–1619. 44:213–223. 33(19):5984–6003. | o 2 no. Biomol J Biophys Chem J | Am J 303

BIOCHEMISTRY