2017-Schubert-Structure of PINK1 in complex wi.pdf

7 D E C E M B E R 2 0 1 7|V O L 5 5 2|N A T U R E|5 1ARTiCLEdoi:10.1038/nature24645Structure of PINK1 in complex with its substrate ubiquitinAlexander F.Schubert1,Christina Gladkova1,Els Pardon2,3,Jane L.Wagstaff1,Stefan M.V.Freund1,Jan Steyaert2,3,Sarah L.Maslen1&David Komander1Parkinsons disease(PD)is a neurodegenerative condition caused by the loss of dopaminergic neurons in the substantia nigra that manifests clinically in the form of characteristic motor defects1.Most cases of PD are sporadic and found in people above the age of 60,but roughly 10%of cases are hereditary forms with an earlier onset.Autosomalrecessive juvenile Parkinsonism(ARJP)is caused by mutations in around 15 PARK genes that have been annotated to date2.The Ser/Thr kinase PINK1 was one of the first PARK genes to be identified3.Mutation of pink1 in Drosophila caused PDlike symptoms,and linked PINK1 to the E3 ubiquitin ligase Parkin 48.Detailed functional reconstruction has since linked PINK1 and Parkin to mitophagy,an important mitochondrial qualitycontrol process911.Mitochondrial damage and subsequent loss of mitochondrial membrane potential leads to the stabilization of PINK1 on the mitochondrial outer membrane(MOM)12 and subsequent dimerization,autophosphorylation and activation of PINK11316.PINK1 activity leads to phosphorylation of ubiquitin on Ser65(hereafter referred to as phosphoubiquitin)1721,and to phosphorylation of the Ubl domain of Parkin on its structurally identical Ser65 residue16,2125.Together,phosphorylation and phosphoubiquitin binding lead to localized Parkin activation on mitochondria,where Parkin ubiquitinates MOM proteins26,triggering a feedforward mechanism21.Mitochondrial ubiquitination recruits mitophagy adaptors and initiates clearance of the damaged mitochondria9,10,27,28.PINK1 is one of the most divergent human protein kinases29.It contains an Nterminal mitochondriatargeting and transmembrane segment,an unconserved region,a kinase domain with three insertions in the N lobe,and a conserved Cterminal region(CTR)of unknown function and structure(Fig.1a,Extended Data Fig.1).Although distinct from other kinases,PINK1 is highly conserved across species(Extended Data Fig.1),which has led to the identification of insect PINK1 orthologues including that of the body louse(Pediculus humanus corporis),PhPINK1.PhPINK1 can be expressed in large quantities in Escherichia coli30.Enabling structural studies of PINK1PINK1 phosphorylates Ser65,which is protected in the ubiquitin fold,and is therefore an unlikely phosphorylation site.Building on previous work on phosphoubiquitin20(Extended Data Fig.2),we have recently shown that wildtype ubiquitin adopts an equilibrium between the common ubiquitin conformation and a distinct second conformation,in which 5strand slippage retracts the C terminus and extends the Ser65containing loop by two residues31(Extended Data Fig.2).This UbCR conformation is low in abundance,but can be stabilized by point mutations including T66V/L67N(hereafter referred to as UbTVLN31,Extended Data Fig.2).Importantly,PhPINK1 binds to UbTVLN with higher affinity than it does to wildtype ubiquitin,and the interface encompasses the extended Ser65 loop31.Consequently,PINK1 has a preference for UbTVLN as a substrate in comparison to the common ubiquitin conformation31.In order to exploit this insight,we raised llama nanobodies(see Methods)against a chemically crosslinked PhPINK1UbTVLN complex(Fig.1b).Nanobody 696(Nb696)did not bind UbTVLN and interacted only weakly with PhPINK1(Extended Data Fig.3a),but formed a gel filtrationstable,noncovalent trimeric complex when both were present(Extended Data Fig.3ac).Also,neither UbTVLN nor Nb696 alone affected PhPINK1 stability,but Nb696 together with UbTVLN increased the melting temperature of PhPINK1 by around 7 C(Extended Data Fig.3d).Structure of a PhPINK1Ub-TVLNnanobody complexWith further optimization we were able to isolate a homogeneous complex comprising residues 143575 of PhPINK1 bound to Nb696 and UbTVLN(Fig.1c,Extended Data Fig.3e,f).Subsequent phosphatase treatment of the complex generated PhPINK1 that was homogeneously phosphorylated at three sites(Extended Data Fig.3g).We crystallized the complex and determined the Xray structure at 3.1 resolution,with welldefined electron density(Figs 1d,e,2,Extended Data Table 1,Extended Data Fig.4a).The structures of Nb696 and UbTVLN closely resemble previously published structures31,32 Autosomal-recessive juvenile Parkinsonism(AR-JP)is caused by mutations in a number of PARK genes,in particular the genes encoding the E3 ubiquitin ligase Parkin(PARK2,also known as PRKN)and its upstream protein kinase PINK1(also known as PARK6).PINK1 phosphorylates both ubiquitin and the ubiquitin-like domain of Parkin on structurally protected Ser65 residues,triggering mitophagy.Here we report a crystal structure of a nanobody-stabilized complex containing Pediculus humanus corporis(Ph)PINK1 bound to ubiquitin in the C-terminally retracted(Ub-CR)conformation.The structure reveals many peculiarities of PINK1,including the architecture of the C-terminal region,and reveals how the N lobe of PINK1 binds ubiquitin via a unique insertion.The flexible Ser65 loop in the Ub-CR conformation contacts the activation segment,facilitating placement of Ser65 in a phosphate-accepting position.The structure also explains how autophosphorylation in the N lobe stabilizes structurally and functionally important insertions,and reveals the molecular basis of AR-JP-causing mutations,some of which disrupt ubiquitin binding.1Medical Research Council Laboratory of Molecular Biology,Francis Crick Avenue,Cambridge CB2 0QH,UK.2VIB-VUB Center for Structural Biology,VIB,1050 Brussels,Belgium.3Structural Biology Brussels,Vrije Universiteit Brussel,1050 Brussels,Belgium.2017 Macmillan Publishers Limited,part of Springer Nature.All rights reserved.ArticlereSeArcH5 2|N A T U R E|V O L 5 5 2|7 D E C E M B E R 2 0 1 7(Extended Data Fig.4b,c).The nanobody binds UbTVLN and both lobes of the kinase domain(Fig.1d,Extended Data Fig.4d,e),which indicates how it could stabilize the complex.Most importantly,the structure reveals how PhPINK1 binds UbTVLN via the N lobe,in particular through insertion 3,and also via the kinase C lobe,where Ser65 of UbTVLN is in a phosphoaccepting position(Fig.1d,e).This suggests that this structure represents a precatalytic state of ubiquitin phosphorylation by PINK1.Unique structural features of PINK1PhPINK1 adopts the wellcharacterized bilobal kinase fold33(Figs 1e,2a,Extended Data Fig.5a).Comparison with an active protein kinase structure such as that of phosphorylase kinase34(Protein Data Bank(PDB)accession number 2PHK,Extended Data Fig.5a,b)reveals highly similar conformations of the catalytically important DFG(DFGin,Asp357,Phe358,Gly359)and HRD(His332,Arg333,Asp334)motifs35,36,formation of an essential GluLys contact in the ATPbinding site(Glu214Lys193 in PhPINK1),and highly similar R and C spines36,confirming that PhPINK1 is in an active conformation(Extended Data Fig.5c).The PhPINK1 structure highlights unique features of PINK1,such as the architecture of the CTR(Fig.2a,b,Extended Data Fig.1).Four additional helices(JM)pack tightly against the C lobe such that the last two helices(L and M)are enveloped by the CTR J and K helices on one side,and the Clobe helices E,H and I on the other side(Fig.2a,b),forming an extended hydrophobic core(Extended Data Fig.5d).From this architecture it is clear how loss of the CTR would affect the activity and stability of PINK130,37.Exposed surface residues of the CTR do not show a high degree of evolutionary conservation(Extended Data Fig.5e),and it is unclear whether and how the CTR contributes to PINK1 function beyond stabilization.The most striking differences between the PhPINK1UbTVLN complex and other kinase structures are located in the kinase N lobe,which contains the three unique insertions,and also features an B helix and an unusually short singleturn C helix36 that harbours Glu214(Fig.2ce,Extended Data Fig.5a,c).Insertion 1(5 amino acids in PhPINK1,34 amino acids in human(h)PINK1,Extended Data Fig.1),which connects strands 2 and 3,is disordered(Fig.2c).Insertion 2(19 amino acids in PhPINK1,21 amino acids in hPINK1)connects the C helix to the 4 strand,and forms a short strand(i,parallel to 4)and an i helix suspended above the BC surface(Fig.2d).Insertion 3(27 amino acids in PhPINK1 and hPINK1)is located between strands 4 and 5,and forms an ordered subdomain that lacks secondary structure,but binds ubiquitin(Figs 1e,2ae,3).This element is the bestconserved insertion in PINK1(Extended Data Fig.1),underscoring its functional importance.Finally,the N lobe contains two conserved phosphorylated residues,Ser202 and Ser204(Fig.2e;Ser228 and Ser230 in hPINK1),which are discussed in Phosphorylation organizes Nlobe insertion.Ub-TVLN binding to PhPINK1The PhPINK1UbTVLN structure has provided an opportunity to characterize a transient kinasesubstrate complex.PhPINK1 binds UbTVLN via a 751 2 interface,and forms bipartite interactions with the N lobe and the activation segment in the C lobe(Fig.3a).The Nlobe part of the interface includes a pocket created by the Glyrich d ahPINK1 PhPINK1PhPINK1 115575PhPINK1 143575i1i2i3MTSCTRKinase N lobeKinase C lobeTMPhPINK1 143575Nb696Ub-TVLNGel fltrationfractioncPhPINK1PhPINK1Cross-linkingPhPINK1PhPINK1Low binding affnityUb-TVLNUb-TVLNUb-TVLNNanobodygenerationNb 696Ub-TVLNXXStable,non-covalenttrimeric complexSelectionbe15957590PhPINK1KDNb696Ub-TVLNCTRUb S654961428381758115611XCTRPhPINK1KDUb-TVLN Ub S65Insertion 3ATP-bindingsiteActivation segmentN lobeC lobeInsertion 3Figure 1|PhPINK1 constructs,nanobody design,and crystal structure of the trimeric PhPINK1Nb696Ub-TVLN complex.a,hPINK1 and PhPINK1 domain architecture.The mitochondrial targeting sequence(MTS),transmembrane domain(TM),insertions 1(i1),2(i2)and 3(i3),and the Cterminal region(CTR)are indicated.b,Nanobodies were raised against the chemically crosslinked PhPINK1UbTVLN complex.c,Nanobody 696(Nb696)forms a stable,noncovalent trimeric complex with PhPINK1 and UbTVLN that can be isolated by gel filtration.Asingle fraction from a gel filtration run is shown.See Extended Data Fig.3f and Supplementary Fig.1 for full and uncropped gels,respectively.d,Crystal structure of the PhPINK1Nb696UbTVLN complex,with PhPINK1 shown under a surface,Nb696 in grey,and UbTVLN in red.The ubiquitinbinding insertion 3 is shown in yellow.Ubiquitin Ser65 is displayed as spheres.See also Extended Data Table 1 and Extended Data Fig.4.e,As d,rotated 90,showing only PhPINK1 and UbTVLN.KD,kinase domain,pink.2017 Macmillan Publishers Limited,part of Springer Nature.All rights reserved.Article reSeArcH7 D E C E M B E R 2 0 1 7|V O L 5 5 2|N A T U R E|5 3loop(residues 164174),insertion 3 and Phe196Tyr198,which engulfs the ubiquitin hairpin residues Ala46Gly47(Fig.3b,Extended Data Fig.6a).Tyr198 is a key residue that forms hydrophobic contacts with Ile44 and especially Val70 of ubiquitin,and a hydrogen bond with Gly47(Fig.3b).Several additional hydrogen bonds occur between insertion 3 and ubiquitin(Fig.3b).The Clobe part of the interface contains the PhPINK1 activation segment(residues 360385),which contacts the extended Ser65containing loop of UbTVLN(Fig.3c).The latter is held in place by polar contacts(linking ubiquitin Glu64 to PhPINK1 Asn383/Ala385,ubiquitin Val66(Thr66 in wildtype ubiquitin)to PhPINK1 Asn383,and ubiquitin His68 to PhPINK1 Asp379).These contacts resemble those reported in kinasesubstrate peptide complexes(Fig.3c,d),and place Ser65 close to a phosphoaccepting position.Flexibility in the Ser65 loop and kinase NtoClobe dynamics would enable catalysis in the presence of nucleotide.We used mutational analysis to validate the ubiquitinbinding interface.A Y198E mutation in PhPINK1 did not affect autophosphorylation(Extended Data Fig.7a),but abolished phosphorylation of UbTVLN and ubiquitin(Fig.3f).Similarly,ubiquitin or UbTVLN with a G47E mutation was no longer phosphorylated(Fig.3f).ARJP mutations in insertion 3(PhPINK1G281D or P268L,Fig.3b,see below)reduced or abrogated kinase activity towards ubiquitin or UbTVLN,and in the case of G281D,also reduced autophosphorylation(Fig.3f,Extended Data Fig.7a).PhPINK1D379A,which prevents contact with His68 on UbTVLN(Fig.3c),reduced phosphorylation of UbTVLN and ubiquitin,but not PhPINK1 autophosphorylation,while the ARJP mutation G382V abrogated kinase activity completely(Fig.3f,Extended Data Fig.7a).This confirms the mode of ubiquitin binding,in line with previous biochemical findings19,22,38(Extended Data Fig.6b).Strikingly,ubiquitin binding seems to be optimized towards the UbCR conformation.The 5strand shift in UbTVLN(Extended Data Fig.2)juxtaposes Val70 and Tyr198(Fig.3b),and allows His68 to contact the activation segment via Asp379(Fig.3c,Extended Data Fig.6c).Superposition of wildtype ubiquitin onto UbTVLN suggests that substratepositioning interactions with the activation segment can be established only with an extended Ser65 loop(Fig.3e,Extended Data Fig.6d).This is consistent with NMR results showing that the Ser65 loop in wildtype ubiquitin does not partake in PhPINK1 contacts31,and is also supported by hydrogendeuterium exchangemass spectrometry(HDXMS)data,which reveal that binding of UbTVLN involves the kinaseactivation segment,while wildtype ubiquitin does not contact the kinase C lobe(Extended Data Fig.6ef).Nevertheless,wildtype ubiquitin binds to PhPINK1 and is phosphorylated,albeit at a roughly 50fold reduced rate31 in comparison to UbTVLN,and NMR data indicate that the proteins interact via Ala46Gly47,Ile44 and Arg4231,which is consistent with HDXMS data(Extended Data Fig.6f).It is possible that the common ubiquitin conformation binds the N lobe and transforms to the UbCR conformation while bound31.This would stabilize the interface and enable interaction with the activation segment and phosphorylation on Ser65.Phosphorylation organizes N-lobe insertionsPINK1 activity is regulated by autophosphorylation13,15,39.Three phosphorylation sites,Thr305,Ser202 and Ser204,resisted phosphatase treatment and were resolved in the crystal structure of active PhPINK1.Thr305 is not conserved(Extended Data Fig.1),and is in an exposed position on the C lobe(Extended Data Fig.7b).By contrast,Ser202 and Ser204(Ser228 and Ser230 in hPINK1)are highly conserved(Extended Data Fig.1),and autophosphorylation of hPINK1 on Ser228 is important for kinase activity13,15.The structure explains the important role of phosphoSer202 and phosphoSer204 in coordinating insertion 3 and insertion 2,respectively(Fig.4a).PhosphoSer204 provides an anchor point for insertion 2 to rest on the BC helix.Similarly,insertion 3 residues Arg282 and Asn283 wrap around phosphoSer202,locking the end of insertion 3 in place(Fig.4a).Hence,while UbTVLN does not contact phosphoSer202 directly,the structural effect of Ser202 phosphorylation on in