Nucleotide-binding mechanisms in pseudokinases

Biosci. Rep. (2016) / 36 / a :e00282 / doi 10.1042/BSR20150226 Nucleo ide-binding mechanisms in pseudokinases Hen ik M. Hamma ´ en*1, Anniina T. Vi anen* and Olli Sil ennoinen*†1 *School o Medicine, Uni e si y o Tampe e, Bioka u 8, FI-33014 Tampe e, Finland †Clinical Hema ology, Depa men o In e nal Medicine, Tampe e Uni e si y Hospi al, Medisiina inka u 3, FI-33520 Tampe e, Finland Synopsis Pseudokinases a e classi ied by he lack o one o se e al o he highly conse ed mo i s in ol ed in nucleo ide (n ) binding o ca aly ic ac i i y o p o ein kinases (PKs). Pseudokinases ep esen ∼10 % o he human kinome and hey a e ound in all e olu iona y classes o kinases. I has become e iden ha pseudokinases, which we e ini ially conside ed somewha peculia dead kinases, a e impo an componen s in se e al signalling cascades. Fu he mo e, se e al pseudokinases ha e been linked o human diseases, pa icula ly cance , which is aising in e es o he apeu ic app oaches owa ds hese p o eins. The ATP-binding pocke is a well-es ablished d ug a ge and elucida ion o he mechanism and p ope ies o n binding in pseudokinases is o signi ican in e es and impo ance. Recen s udies ha e demons a ed ha membe s o he pseudokinase amily a e e y di e se in s uc u e as well as in hei abili y and mechanism o bind n s o pe o m phospho yl ans e eac ions. This di e si y also p ecludes p edic ion o pseudokinase unc ion, o he impo ance o n binding o said unc ion, based on p ima y sequence alone. Cu en ly a ailable da a indica e ha ∼40% o pseudokinases a e able o bind n s, whe eas only ew a e able o ca alyse occasional phospho yl ans e . Pseudokinases employ di e se mechanisms o bind n s, which usually occu s a low, bu physiological, a ini y. ATP binding se es o en a s uc u al ole bu in mos cases he unc ional oles a e no p ecisely known. In he p esen e iew, we discuss he a ious mechanisms ha pseudokinases employ o n binding and how his o en low-a ini y binding can be accu a ely analysed. Key wo ds: ATP, kinase ac i i y, kinome, nucleo ide binding, pseudokinase, signalling. Ci e his a icle as: Bioscience Repo s (2016) 36, e00282, doi:10.1042/BSR20150226 INTRODUCTION In hei landma k pape in 2002, Manning and colleagues p esen- ed o he i s ime a comp ehensi e ca alogue o human p o ein kinases (PKs) [1]. One o he salien indings o hei analysis was ha ∼10% o he 518 human PKs lack a leas one o he classical conse ed ca aly ic esidues desc ibed by Hanks e al. [2]: he β3 lysine (K in he ‘VAIK’ consensus mo i , Figu e 1), he ca aly ic aspa a e (D in ‘HRD’) o he ca ion-binding as- pa a e (D in ‘DFG’). Some o hese kinases, like he WNK (‘Wi h no lysine’) amily, we e al eady known o ha e phos- pho yl ans e ac i i y despi e he lack o canonical ca aly ic ............................................................................................................................................................................................................................................................................................................ Abb e ia ions: ADCK, aa F domain-con aining p o ein kinase; AMP-PNP, adenyla e-imidodiphospha e; ANK, anky in epea domain (in RNase L); ANPa/ANPb, a ial na iu e ic pep ide ecep o ype A/B, also known as a ial na iu e ic pep ide ecep o 1 (NPR1) and NPR2, espec i ely. Also known as GC-A/GC-B; ATPγS, adenosine 5-[γ- hio] iphospha e; BAK1, BCL2-an agonis /Kille 1; BIR2, BAK1-in e ac ing ecep o -like kinase 2; BSK8, b assinos e oid signalling kinase 8; CaM-kinase, Ca2+/calmodulin-dependen p o ein kinase; CASK, calcium/calmodulin-dependen se ine p o ein kinase; COP1, cons i u i e pho omo phogenesis p o ein 1; EGFR, epide mal g ow h ac o ecep o ; ePK, euka yo ic p o ein kinase; GC, guanyla e cyclase; GUCY2, guanyla e cyclase, e.g. GUCY2C, also known as GC-C, and hea s able en e o oxin ecep o (HSER); HER3, human epide mal g ow h ac o 3, also known as ERBB3; IRAK, in e leukin-1 ecep o -associa ed kinase; ITC, iso he mal i a ion calo ime y; JAK, Janus kinase; JH, JAK homology; LKB1, li e kinase B1; MLKL, mixed lineage kinase domain-like; n , nucleo ide; NTE, N- e minal ex ension; PAN3, PAB1P-dependen poly(A)-nuclease; PK, p o ein kinase; PDZ, p o ein domain ound in PSD95, Dlg1 and zo-1; PKA, cAMP-dependen p o ein kinase A; PKD, pseudokinase domain; PKL, p o ein kinase-like; RIP3, ecep o -in e ac ing se ine- h eonine kinase 3; RLK, ecep o -like kinase; ROPK, hop y kinase; ROR, ecep o y osine kinase-like o phan ecep o ; RTK, ecep o y osine kinase; SH3, S c-homology 3 domain; SPR, su ace plasmon esonance; STRAD, S e20- ela ed adap o ; TK, i in kinase; TLR, Toll-like ecep o ; TSA, he mal shi assay; VRK, accinia- ela ed kinase; WNK, Wi h no lysine. 1Co espondence may be add essed o ei he o hese au ho s (email [email p o ec ed] o [email p o ec ed]). si es. Ye , he o he 50 iden i ied p o eins o p o ein domains we e deemed likely o be inac i e and consequen ly dubbed ‘pseudok- inases’. Subsequen ly, also some o hese pseudokinases (like Haspin) we e shown o be ca aly ically ac i e, and hus could be eclassi ied as a ypical PKs [3]. Fo hese and some mo e ecen examples o ca aly ic ac i i y like KSR2 [4], HER3 [5], JAK2 JH2 [6]o CASK[7] he dis inc ion o pseudokinase and a ypical kinase has become somewha unclea . Howe e , as p o- posed by Eye s and Mu phy [8], he bioin o ma ic de ini ion o a pseudokinase (wi h he inclusion o kinases ha ha e expe i- men ally been ound o be inac i e) should be main ained o he sake o cla i y, and will be used in he p esen e iew as well. c 2016 Au ho s. This is an open access a icle published by Po land P ess Limi ed and dis ibu ed unde he C ea i e Commons A ibu ion Licence 3.0. 1 H.M. Hamma ´ en, A.T. Vi anen and O. Sil ennoinen Figu e 1 Conse ed mo i s and esidues con ibu ing o n binding and kinase ac i i y in (pseudo)kinases (A) Schema ic depic ion o he seconda y s uc u e elemen s o ePKs. Rela i e sizes and posi ions o elemen s a e based on PKA (PDB: 4WB5). (B) Sequence alignmen o selec ed pseudokinases and PKL p o eins classi ied based on cu en in o ma ion on n binding. Conse ed PK egions ele an o n binding a e shown. The six highly conse ed esidues con ibu ing di ec ly o n binding o ca aly ic ac i i y a e highligh ed. *The ga ekeepe esidue is no pa o he 10 conse ed kinase esidues iden i ied in [14]. All sequences ep esen human p o eins unless o he wise no ed. The sequence alignmen was made using Clus al W [137,138] and manually co ec ed based on c ys al s uc u es and p e ious alignmen s [32,92,139] whe e a ailable. Fo non-ePKs he sequences shown a e he (p edic ed) unc ional/s uc u al equi alen s o he conse ed esidue in ques ion (seconda y s uc u e o e.g. ADCK3 o Fam20 kinases is di e en om he one shown in A). Fam20C is included as an example o an ac i e Fam20 kinase. (C) 3D s uc u e o human PKA (PDB: 4WB5) shown as an example o an a che ypal ePK. ATP is shown in s icks and he wo magnesium ca ions as pu ple sphe es. Colou s o seconda y s uc u e elemen s a e as in (A). Pseudokinases a e sp ead h oughou all PK amilies [1], in- dica ing ha a leas mos o hem ha e eme ged epea edly om ac i e kinases h oughou e olu ion. Despi e he ca aly ically in- ac i e na u e o mos pseudokinases s udied hus a , a ecen su ey ound ha 13 ou o he 31 di e se pseudokinases s udied a e s ill able o bind n s [9]. Fu he s udies on he cha ac e is ics and unc ions o n binding in pseudokinases ha e shed ligh on his unexpec ed inding, and shown ha pseudokinases employ di e se mechanisms o bind ATP and sugges ha n binding can play an impo an s uc u al and unc ional ole. Se e al excellen e iews on he ad ances in he pseudok- inase ield ha e been w i en [3,8,10], including he po en ial impo ance o pseudokinases in human disease [11–13]. Recen moun ing e idence o he impo ance o he n -binding si e o pseudokinases in pa icula , howe e , wa an s a ocused discus- sion on his aspec . The p esen e iew aims o p o ide such a comp ehensi e upda e on he he e ogenei y o n binding in pseudokinases. MOTIFS INVOLVED IN NT BINDING IN PK DOMAINS Pseudokinases belong o he p o ein kinase-like (PKL) supe - amily which sha e a conse ed old wi h an N lobe comp ised mos ly o βs ands and a mos ly α-helical C lobe (Figu e 1C). The ca aly ic mechanism in PKL p o eins in ol es 10 esidues [14] ha a e highly conse ed e en i he p o eins may o he wise .......................................................................................................................................................................................................................................................................................................................................................................... 2c 2016 Au ho s. This is an open access a icle published by Po land P ess Limi ed and dis ibu ed unde he C ea i e Commons A ibu ion Licence 3.0. Nucleo ide binding in pseudokinases lack sequence conse a ion [14,15]. Six ou o hese 10 esidues a e di ec ly in ol ed in n o subs a e binding o ca alysis [14] (highligh ed in Figu e 1B), whe eas he unc ion o he emain- ing ou esidues is s ill no comple ely unde s ood [14], bu is p obably mainly s uc u al [16]. The i s o he c i ical esidues o n binding is Gly52 (as he de ac o a che ypal p o ein kinase, human cAMP-dependen p o ein kinase A (PKA) is usually used o numbe ing o amino acid esidues in p o ein kinase mo i s and will also be used in he p esen e iew) in he glycine- ich loop (‘Gly- ich loop’, also known as he ‘phospha e-binding loop’ o ‘P-loop’) loca ed be ween s ands β1andβ2(Figu e 1). The unc ion o he Gly- ich loop is bes unde s ood in PKA, in which glycine a his posi ion enables he pep ide backbone o he ip o he loop (Se 53) obind heγ-phospha e o ATP. Mu a ion o he Gly- ich loop, and especially Gly52, lowe s a ini y owa ds ATP and a ec s kinase ac i i y [17]as heγ-phospha e can no longe be e icien ly posi ioned o phospho yl ans e be ween he ip o he Gly- ich loop and a basic esidue (Lys168) om he so-called ‘ca aly ic loop’ si ua ed be ween β6andβ7(Figu e 1)[18]. Al hough he unc ion o he Gly- ich loop is well known o PKA, he mo i has no been ex ensi ely s udied in o he kinases and i is unclea how uni e sal his unc ion is. The second esidue is Lys72 o he ‘VAIK’ mo i in β3, which is he mos conse ed esidue in all PKL p o eins, and he only esidue no missing in any o he known amilies (Figu e 1)[14]. Despi e i s i ually uni e sal conse a ion and i s posi ion nex o he αand βphospha es o ATP (Figu e 2, PKA), he unc ion o Lys72 in n binding is no en i ely clea , and i has been epo ed o be dispensable o n binding in mul iple canonical kinases [19– 22]. Howe e , he lysine seems o be equi ed o n binding in he pseudokinases GUCY2C [23], HER3 [5], TRIB2 [24] and mu ine (and o a lesse ex en also human) MLKL [25,26]. Lys72 (o ano he lysine in i s spa ial posi ion, like Lys223, WNK1 in he WNK amily [27]) is absolu ely equi ed o ca aly ic ac i i y bo h in mul iple canonical kinases [19,20,22], as well as in he low- ac i i y pseudokinase JAK2 JH2 [6]. E en hough i s unc ion in ATP binding is somewha unclea , Lys72 is c i ical in making a sal b idge o he conse ed Glu91 in he C helix (αC) (Figu e 2, PKA), hus linking αC o he n -binding pocke and he helix in he ‘in’ posi ion, which is a hallma k o he ac i e kinase con o ma ion [16,28]. The las h ee o he six conse ed esidues a e equi ed o ca alysis: Asp166 (‘HRD’) and Asn171 in he ca aly ic loop, and Asp184 in he ‘DFG’ mo i (Figu e 1). Asn171 and Asp184 pa i- cipa e in he binding o he wo di alen ca ions accompanying ATP in he canonical mode o ATP binding in kinases (Figu e 2, PKA). Simila ly o he β3 lysine, Asp166 and Asp184 a e no ab- solu ely equi ed o ATP binding, bu a he needed o e icien ca alysis [22], Asp166 being he ca aly ic base in he phospho yl ans e eac ion [29]. In addi ion o hese conse ed single esidues, he pu ine pocke o he n -binding si e is lined wi h a g oup o hyd o- phobic esidues om β2(Val 57)andβ3(Ala 70) om he N lobe, and β7 (Leu173) om he C lobe (Figu e 2, PKA). These esidues a e pa o he so-called ‘ca aly ic spine’ (C spine), which is a conse ed hyd ophobic s uc u e ypically ound in ac i e kinases [16,30,31], and which is comple ed upon binding o a n ’s pu ine ing be ween he N and C lobes (shown in ligh blue in Figu e 2). Addi ionally, PKs also ha e ano he nonlinea , conse ed s uc- u al elemen called he ‘ egula o y spine’ (R spine) [16,30,31], made up o ou esidues om DFG, HRD, αC and he αC-β4 loop (shown in beige in Figu e 2). This s uc u e is s abilized by phospho yla ion o he ac i a ion loop in canonical kinases, and a ully assembled R spine is usually a p e equisi e o kinase ac i i y [16,30,31]. Func ional p edic ion based on sequences – excep ions o he ule The a o emen ioned conse ed esidues ha e been used o p e- dic n -binding abili y and/o ca aly ic ac i i y o unknown PKs. The p esence o an in ac Gly- ich loop and VAIK mo i s, o ex- ample, seem o ha e been he bes p edic o s o ca aly ic ac i i y in he pas [32] – e en in noncanonical ac i e kinases like CASK, which lacks Asp184 bu has an in ac Gly- ich loop and a β3lysine. Howe e , n -binding abili y and mechanisms canno be eliably p edic ed om sequence da a alone, as is shown by he inno a i e non-canonical n -binding modes employed by se e al pseudok- inases (see below). Thus, accu a e biochemical and biophysical measu emen s ha allow ca e ul di e en ia ion be ween n bind- ing, ca aly ic ac i i y, and hei possible physiological oles, a e a necessi y when analysing di e gen p o eins like pseudokinases. METHODOLOGY OF MEASURING NT BINDING The inhe en p ope ies o pseudokinases, i.e. low o absen kinase ac i i y coupled o unknown n binding, a e o en incom- pa ible wi h adi ional kinase assays and o en mul iple me hods and igo ous con ols need o be employed in o de o ob ain eli- able esul s. Cu en echniques, and hei challenges wi h espec o pseudokinases ha e been ecen ly desc ibed in he excellen e iew by Luce e al. [33]. In o de o p o ide he backg ound o he ollowing inspec ion o n -binding p ope ies in pseudok- inases, we will highligh some impo an echnical aspec s in he p esen s udy as well. Phospho yl ans e o ATP hyd olysis ac i i y o pseudok- inases is gene ally o de s o magni ude lowe compa ed wi h ac i e kinases, and he e o e e en iny kinase con amina ions can lead o alse conclusion o pseudokinase ca aly ic ac i i y [34]. As an example, in e eb a e mi o ic checkpoin p o ein BUBR1 kinase-inac i a ing mu a ions and dele ion s udies demons a ed ha he ini ially obse ed kinase ac i i y was ac ually de i ed om con amina ing kinases [35]. Fu he mo e, as wi h con en- ional kinases, choice o subs a e can be c i ical, and he lack o phospho yla ion o ap io ilikely subs a e candida es o an a i icial subs a e does no p eclude ha he p o ein could ha e .......................................................................................................................................................................................................................................................................................................................................................................... c 2016 Au ho s. This is an open access a icle published by Po land P ess Limi ed and dis ibu ed unde he C ea i e Commons A ibu ion Licence 3.0. 3 H.M. Hamma ´ en, A.T. Vi anen and O. Sil ennoinen Figu e 2 Di e se ATP-binding pocke s and n -binding modes among pseudokinases C ys al s uc u es o ATP-binding pocke s o selec ed ep esen a i e (pseudo)kinases and PKL p o eins wi h a ying n -binding modes. Shown a e human PKA (PDB: 4WB5), human RNase L (4OAV), human JAK2 JH2 (4FVQ), A abidopsis haliana BSK8 (4I94), human STRADα(3GNI), human VRK3 (2JII), A. haliana BIR2 (4L68), Ra us no egicus WNK1 (4Q2A), human MLKL (4MWI), human ROR2 (4GT4), human TRIB1 (5CEM) and human ADCK3 (4PED). ATP shown in WNK1, MLKL and ADCK3 was modelled based on PKA (4WB5), as no ATP-bound s uc u es exis , e en hough hey e i iably bind .......................................................................................................................................................................................................................................................................................................................................................................... 4c 2016 Au ho s. This is an open access a icle published by Po land P ess Limi ed and dis ibu ed unde he C ea i e Commons A ibu ion Licence 3.0. Nucleo ide binding in pseudokinases speci ic ac i i y in he con ex o a physiological p o ein complex [8,12]. X- ay c ys allog aphy gi es a omic-le el in o ma ion on he s uc u e o a n -binding pocke and can be used o p edic , o in he case o n -bound s uc u es, unequi ocally e i y n binding. KSR2 [4], HER3 [5,36], TYK2 JH2 [37], JAK2 JH2 [38], STE20- ela ed adap o alpha (STRADα)[39], ILK [40]andCASK[7], as well as a ew o he pseudokinases and PKLs om human and o he species, ha e been c ys allized in complex wi h n s p o id- ing solid p oo and mechanis ic in o ma ion o n binding (see Table 1). While X- ay c ys allog aphy is limi ed o de e mina ion o igid p o ein s uc u es, NMR is applicable o small p o eins (M ⩽30–40 kDa [41]) in a soluble s a e o e eal dynamic s uc- u es [42], and e.g. e i y absence o p esence o n binding [43]. Assays measu ing n binding a e o en less sensi i e o low le els o con amina ions han kinase assays. Iso he mal i a- ion calo ime y (ITC) and su ace plasmon esonance (SPR) a e quan i a i e, label- ee me hods o de ec ion o n binding. ITC is he only me hod o di ec measu emen o he modynamic pa ame e s including en halpy, Kdand s oichiome y o ligand– p o ein in e ac ion, as i di ec ly measu es he abso bed o emi ed hea du ing a (bio)molecula in e ac ion [34]. SPR, on he o he hand, p o ides di ec in o ma ion abou binding kine ics and a - ini y h ough an op ical assay [37,44]. A ini y (and o a lesse ex en kine ics) es ima es, can also be ob ained using spec o luo ome ic assays using in insic luo escence measu emen s o luo escen ly agged n s. The in- insic luo escence me hod depends on yp ophan, y osine and phenylalanine esidue(s), whose spec oscopic cha ac e - is ics change in esponse o n binding, which makes hese assays somewha limi ed in hei applicabili y. Mo e widely- used luo escence applica ions use luo escen n analogues in luo escence spec oscopy o luo escence pola iza ion assays. Man (2(3)-O-(N-me hylan h aniloyl)) [5,6,45]andTNP(2 -3- O-(2,4,6- ini ophenyl)) [7,46] a e commonly used labels, ha show enhancemen in luo escence once bound o he hyd o- phobic ATP-binding pocke o a (pseudo)kinase. Fluo escence pola iza ion (also known as luo escence aniso opy) assays, us- ing e.g. he long-wa eleng h luo escen ly-labelled BODIPY– ATP-γ-S n analogue, measu e he inc ease in luo escence po- la iza ion upon binding o a labelled small-molecule ligand o a p o ein. Due o i s sensi i i y, he assay is well-sui ed o sc een- ing pu poses, bu i has also been used o assess binding o n analogues o pseudokinases [47]. Me hods using agged n s al- low es ima ion o he Kd o he n analogues, bu non-speci ic binding is possible. Values o unlabelled n s can be es ima ed by hei abili y o displace he luo escen analogues, which is im- po an , as he p esence o luo escen ags can change he binding p ope ies o a n [39]. Fo compe i ion expe imen s he meas- u emen window o he assay is, howe e , inhe en ly es ic ed by he binding a ini y o he p obe in ques ion. Binding o n s (o indeed any ligand) o p o eins can also be analysed using he mal shi assays (TSA), which measu e changes in he mal s abili y o a p o ein upon binding o ligands. Changes in he mal s abili y a e measu ed by obse ing hea - induced un olding o he p o ein using, e.g. in insic luo escence o a p o ein [24,34] o ex insic luo escence o a dye [9]asa eadou . Ligand binding gene ally s abilizes p o ein s uc u es, and esul s in an inc eased mel ing empe a u e (Tm). Co ela ion be ween Tmand Kdo IC50 is dependen on he p o ein–ligand pai in ques ion, and TSAs should no be used as a sole me hod o Kdde e mina ion [48,49]. Fu he mo e, o low-a ini y binde s o cases whe e ligand binding does no cause la ge Tmshi s (Tm), sensi i i y can be a p oblem. The me hods desc ibed abo e a e p ima ily sui ed o pu i ied ecombinan p o eins. N a ini y ch oma og aphy is one o he ew ools o iden i ica ion o n binde s also om cell lysa es. The me hod uses a ini y beads o immobilized ATP o cap u e n -binding p o eins and de ec ion by, e.g. Wes e n blo ing [23] o mass spec ome y in a high- h oughpu app oach [50]. In summa y, es ima es o n -binding a ini y be ween di e en me hods can a y qui e signi ican ly, and he choice o me hod has o be o en de e mined empi ically. In gene al, label- ee me hods, like ITC, should be a ou ed o accu acy and p ecision, bu due o limi a ions in, e.g. p o ein amoun , o he assays a e also widely used. Fu he mo e, he unc ional ole and ele ance o n binding can a ely be assessed using ecombinan p o eins alone, and usually equi es si e-di ec ed mu agenesis in cellula assays. Addi ionally, he modes o n binding a y widely in pseudokinases, and hey can only be e ealed by sol ing c ys al o NMR s uc u es. MODES OF NT BINDING IN PSEUDOKINASES Gi en he la ge a ia ion o pseudokinase sequences especially a o nea he n -binding si e (Figu e 1), i is no su p ising ha pseudokinases display a wide a ie y o di e en n -binding mechanisms. The mechanism o ATP binding is cu en ly un- known in mos pseudokinases, bu s uc u al in o ma ion o 21 pseudokinases (Table 1) has shown ha pseudokinases can bind Figu e 2 Con inued. adenine n s. ATP o ATP-analogues (e.g. AMP-PNP o BSK8) a e shown as s icks wi h elemen s colou ed as ollows: ca bon: black, oxygen: ed, ni ogen: blue, phospho us: o ange. Di alen ca ions a e shown as pu ple sphe es. The halide ion in WNK1 is shown in g een. The R spine is shown as a beige olume illing model, whe eas he op o he C spine, encompassing he hyd ophobic pu ine-binding pocke is shown in ligh blue. Hyd ophobic side chains occluding he pu ine-binding pocke a e shown as pa o he C spine o VRK3, BIR2 and ROR2, whe e he pocke is occluded. Gly- ich loop glycines a e shown as g ey sphe es wi h Gly- ich loop side chains omi ed, unless o special no e. Wa e molecules om he c ys al s uc u es ha e been omi ed o cla i y. *Only one possible con o ma ion gi en o Gln486, ADCK3 is shown. .......................................................................................................................................................................................................................................................................................................................................................................... c 2016 Au ho s. This is an open access a icle published by Po land P ess Limi ed and dis ibu ed unde he C ea i e Commons A ibu ion Licence 3.0. 5 H.M. Hamma ´ en, A.T. Vi anen and O. Sil ennoinen Table 1 Summa y o published pseudokinase c ys al s uc u es Abb e ia ions: P-PCP, β,γ-me hyleneadenosine 5- iphospha e; AMP-PN, AMP phospho amida e. *C ys allized wi h su ace mu a ions (W659A, W777A, F794H). †In e ed om biochemical da a o close homology o published s uc u es showing he n -binding mode. ‡Can be made ca ion-dependen wi h ou poin mu a ions [83]. §Mu a ed as explained in [77]. ¶No iden ical wi h ROP2 sequence in 2W1Z. C ys al s uc u es P o ein PDB ID Species Ligand Complex Kd o ATP Binding mode O he KSR2 2Y4I Homo sapiens ATP +Mg MEK1 Unknown 1 ca ion Ac i e, phospho yla es MEK1 [4] HER3 4OTW H. sapiens Bosu inib – 10−6M[5] 1 ca ion Ac i e, au ophos- pho yla es i s own in acellula egion, when immobilized on esicles [5] 4RIW H. sapiens AMP-PNP + Mg EGFR KD (V624R, F973A, L977A) 4RIX (Q709R) H. sapiens AMP-PNP + Mg EGFR KD (V624R, F973A, L977A) 4RIY (E909G) H. sapiens AMP-PNP + Mg EGFR KD (V624R, F973A, L977A) 3KEX H. sapiens AMP-PNP + Mg – 3LMG H. sapiens AMP-PNP + Mg – TYK2 JH2 3ZON H. sapiens IKK1 – 10−5M[37] 1 ca ion Inac i e [37] 4WOV H. sapiens BMS-066 – 4OLI H. sapiens Inhibi o 7012 JH2-JH1 (D1023N) 5C03 H. sapiens ATPγS+Mg – 5C01 H. sapiens Py azine inhibi o – JAK2 JH2 4FVP* H. sapiens –– 10 −6M[6,45] 1 ca ion Ac i e, au ophos- pho yla es on S523 and Y570 [6] 4FVQ* H. sapiens ATP +Mg – 4FVR (V617F)* H. sapiens ATP +Mg – JAK1 JH2 4L00 H. sapiens –– 10 −6M[45] 1 ca ion†Inac i e [65] 4L01 (V658F) H. sapiens –– MLKL 4BTF Mus musculus –– 10 −5M (TSA) [26] No ca ion† [9,25,26] Inac i e [25] 4MWI H. sapiens –– 4M67 H. sapiens –– 4M68 M. musculus –– 4M69 M. musculus –RIP3KD STRADα3GNI H. sapiens ATP MO25 10 −4–10−6M [39,82] No ca ion [9] Inac i e [140] 2WTK H. sapiens AMP-PNP MO25 +LKB1 .......................................................................................................................................................................................................................................................................................................................................................................... 6c 2016 Au ho s. This is an open access a icle published by Po land P ess Limi ed and dis ibu ed unde he C ea i e Commons A ibu ion Licence 3.0. Nucleo ide binding in pseudokinases Table 1 Con inued. C ys al s uc u es P o ein PDB ID Species Ligand Complex Kd o ATP Binding mode O he VRK3 2JII H. sapiens – – None N/A Inac i e ILK 3KMW H. sapiens ATP +Mg α-Pa in 10−6M[34] 1 ca ion Inac i e [34,40] 3KMU H. sapiens –α-Pa in 3REP H. sapiens ATP +Mn α-Pa in CASK 3C0G H. sapiens 3-AMP – 10−3M[7,9] No ca ion Ac i e, ca ions inhibi binding and ac i i y. [7,24,83] 3C0I H. sapiens 3-AMP – 3C0H H. sapiens AMP-PNP (only AMP isible) – 3TAC H. sapiens – Lip in-α2 3MFS (4M)‡H. sapiens AMP-PNP – 3MFU (4M)‡H. sapiens AMP-PNP + Mn – 3MFT (4M)‡H. sapiens –– 3MFR (4M)‡H. sapiens –– ROR2 3ZZW H. sapiens – – None [55] N/A Inac i e [55,102] 4GT4 H. sapiens –– BIR2 4L68 A abidopsis haliana – – None [94] N/A Inac i e [94] BSK8 4I92 A. haliana – – Unknown 1 ca ion Inac i e [76] 4I93 A. haliana –– 4I94 A. haliana AMP-PNP – Ti in 4JNW H. sapiens –– 10 −4M[113] Unknown Dispu ed [112–114] 1TKI H. sapiens –– PAN3 4CYI Chae omium he mophi- lum ATP +Mg – Unknown, bu p obably a he high a ini y [78] 1 ca ion Unknown, physiological ole o PKD is o shu le poly ibo- nucleo ides o PAN2 [81] 4CYJ C. he mophi- lum ATP +Mg PAN2 4CZY Neu ospo a c assa AMP-PNP + Mg PAN2 4BWK N. c assa ATPγS– 4BWX§N. c assa ATPγS+Mg 4BWP D osophila melano- gas e AMP-PN – 4XR7 Saccha omyces ce e isiae – PAN2 ROP2 2W1Z T. gondii – – None [43] N/A Inac i e [43] 3DZO¶T. gondii Mg – ROP5B 3Q5Z T. gondii – – Unknown 2 ca ions Inac i e [110] 3Q60 T. gondii ATP +Mg – 4LV5 T. gondii ADP IRGa6 (M. musculus) .......................................................................................................................................................................................................................................................................................................................................................................... c 2016 Au ho s. This is an open access a icle published by Po land P ess Limi ed and dis ibu ed unde he C ea i e Commons A ibu ion Licence 3.0. 7 H.M. Hamma ´ en, A.T. Vi anen and O. Sil ennoinen Table 1 Con inued. C ys al s uc u es P o ein PDB ID Species Ligand Complex Kd o ATP Binding mode O he ROP5C 4LV8 T. gondii ADP +Mg IRGa6 (M. musculus) Unknown 1 ca ion Unknown ROP8 3BYV T. gondii Mg – Unknown Unknown Unknown RNase L 4O1O Sus sc o a –2-5A10 −3M[53] 2 ca ions Inac i e [52–54] 4O1P S. sc o a AMP-PNP + Mg 2-5A 4OAU H. sapiens ADP +Mg 2-5A 4OAV H. sapiens AMP-PCP +Mg RNA +pUp WNK1 4Q2A Ra us no egicus B – Unknown Unknown Ac i e [124] 3FPQ R. no egicus –– 4PWN H. sapiens –– M iN 3OTV Mycobac e ium ube cu- losis – – None [47] Unknown Inac i e [47] 3OUK M. ube cu- losis –– 3OUN M. ube cu- losis – FhaA 3UQC M. ube cu- losis –– ADCK3 4PED H. sapiens – – Binds p e e en ially ADP [108] Unknown Inac i e, can be ac i a ed wi h a single Gly- ich loop mu a ion [108] ATP in a canonical ashion employing wo ca ions o , as in mos cases, in a non-canonical ashion wi h one o no ca ions isible in he c ys al s uc u e. In addi ion, some pseudokinases do no bind n s a all, and ob ain ei he an ac i e o inac i e con o ma ion by amino acid subs i u ions and s uc u al ea angemen s. Finally, a ecen analysis o 31 pseudokinases showed ha some pseudok- inases we e s abilized by he p esence o only ca ions wi hou n s [9], bu he signi icance o his inding is cu en ly unclea . In he p esen s udy, we p o ide a comp ehensi e s uc u al iew on n binding in pseudokinases wi h a ocus on he n -binding mode in pseudokinases o which s uc u al da a a e cu en ly a ailable. Bind n s PKA PKA ep esen he a che ype o a canonical PK [51], and i s mode o n binding is he mos p e alen among known PKL membe s. PKA binds i s n ligand ATP wi h wo di alen ca ions posi ioned be ween he phospha es using he c i ical esidues desc ibed abo e (Figu e 2). RNase L The only known pseudokinase o da e displaying a canonical n -binding mode is he mammalian endo ibonuclease RNase L. RNase L unc ions in he ype I in e e on (IFN) esponse whe e i is ac i a ed by 2,5-oligoadenyla e (2-5A) second messenge s and clea es in acellula RNA. RNase L consis s o an anky in epea (ANK), a PK and a RNase domain. Despi e i s ini ial classi ica ion as an ac i e kinase [1], he cu en consensus is ha RNase L is an inac i e pseudokinase [52–54], as i does no au ophospho yla e [52], o phospho yla e he gene ic sub- s a e myelin basic p o ein (MBP) [54]. The PK in RNase L is hough o unc ion as a sca old o homodime iza ion [54]. RNase L binds ATP wi h mic omola a ini y and ATP binding ac i a es RNase ac i i y bo h in i o and in cells, bu is dis- pensable o RNase L dime iza ion in he p esence o 2-5A [53]. The wo ecen ly published c ys al s uc u es o human and po - cine n -bound RNase L [53,54] show a PK old wi h comple e R and C spines esembling he adi ional ac i e kinase con- o ma ion (Figu e 2)[31]. The s uc u es, howe e , also show a non-canonical C lobe wi h subs i u ions in he ac i a ion loop and subs a e ecogni ion si es, which p obably accoun o he inac i i y o RNase L [52]. Bo h sol ed s uc u es show a ully .......................................................................................................................................................................................................................................................................................................................................................................... 8c 2016 Au ho s. This is an open access a icle published by Po land P ess Limi ed and dis ibu ed unde he C ea i e Commons A ibu ion Licence 3.0. Nucleo ide binding in pseudokinases o de ed n in he ATP-binding pocke , which esembles RNase L’s closes ac i e homologue inosi ol- equi ing p o ein 1 (IRE1). N binding is coo dina ed by conse ed PK esidues, excep one uncommon subs i u ion a Gly186 (Asp505, RNase L), which o ms an addi ional in e ac ion o one o he ca ions (Figu e 2). The binding pocke is also lanked by he s uc u ed PK-ANK linke , which pa icipa es in 2-5A binding and in e domain in e ac ions [53,54]. HER3 The mos common n -binding mode among he known pseudok- inase s uc u es is a one-ca ion binding mode i s seen in hu- man epide mal g ow h ac o amily pseudokinase HER3 (also known as ERBB3). As wi h o he kinase amilies, pseudokinases can also be ound in he g oup o 58 human ecep o y osine kinases (RTKs), eigh o which ha e been sugges ed o con- ain in acellula pseudokinase domains (PKDs) [55]. Clinically he mos p ominen o hese is HER3, which media es cellu- la signalling h ough ligand-induced he e odime iza ion wi h epide mal g ow h ac o ecep o (EGFR) and HER2 and al- los e ic ac i a ion o hei kinase ac i i y [36]. I s clinical el- e ance is exace ba ed by he ac ha HER3-media ed HER2- ac i a ion can cause esis ance o HER2- a ge ing cance he - apies [56]. The mos no iceable ea u es o he kinase domain o HER3 a e he lack o Glu91 (subs i u ed by His740, HER3, num- be ing as in PDB: 4RIW) and Asp166 (Asn815, HER3), he la e o which leading o he classi ica ion o HER as a pseudokinase [1,3]. The domain is, howe e , able o bind ATP igh ly wi h a Kdo ∼1μM[5], and se e al c ys al s uc u es wi h he ATP- analogue adenyla e-imidodiphospha e (AMP-PNP) ha e been e- po ed (Table 1)[5,36,57], all o which show n -bound HER3 in an inac i e αC-ou con o ma ion. The n binds oge he wi h a single Mg2+ca ion coo dina ed be ween he AMP-PNP phos- pha es, Asn171 (Asn820, HER3)andAsp 184 (Asp833, HER3). The con- se ed β3 lysine (Lys723, HER3) binds o bo h he αphospha e and Asp833, HER3 in a non-canonical manne mos likely due o he lack o a Glu91-equi alen in αC. In e es ingly, HER3 shows ca aly ic ans-au ophospho yla ion ac i i y in i o, when immobilized on esicles a high local concen a ions [5]. The biological ele ance o his is unce ain, howe e [10,55], as he weak kinase ac i i y has been epo ed o be dispensable a leas o HER3-media ed ligand-dependen signalling [55]. JAK JH2 Janus kinases (JAKs) a e non-RTKs wi h a a e domain a chi- ec u e con aining a pseudokinase (JAK homology 2, JH2) and a kinase domain (JH1) in andem. JH2 media es impo an eg- ula o y unc ions and is needed bo h o supp ession and ull ac i a ion o JH1 ac i i y [58–62]. JAKs unc ion in a my iad o c i ical biological p ocesses anging om egula ion o he immune sys em o haema opoiesis and me abolism [63]. The iden i ica ion o mul iple disease d i e mu a ions in JAK JH2s ha e made hem p obably he mos clinically s udied g oup o pseudokinases o da e. The mos p ominen o hese mu a ions is he V617F mu a ion in JAK2 JH2 unde lying mul iple myelop o- li e a i e neoplasms [64]. While JAK2 JH2 has been shown o possess low au o egula o y kinase ac i i y [6], JAK1 and TYK2 JH2s a e p obably ca aly ically inac i e pseudokinases as no au o- phospho yla ion o kinase ac i i y owa ds exogenous subs a es has been de ec ed [37,65]. Th ee o he ou JAK JH2 domain s uc u es ha e been sol ed o e he las ew yea s. The JH2s in all h ee (JAK1, JAK2, TYK2) a e e y simila and show a kinase domain s uc u e wi h an ac- cessible n -binding si e, a pa ially deg aded Gly- ich loop (Gly50 and Gly52 a e p esen , howe e ) and an abno mally sho ac i - a ion loop [37,38,65]. The s uc u es esemble he HER3 PKD in hei n -binding si e a chi ec u e, as he canonical Lys72–Glu91 bond is eplaced wi h a Lys72–Asp184 bond and an aspa agine sub- s i u es o Asp166 in he ca aly ic loop (Figu e 1). In JAK2 JH2, o example, he n phospha es coo dina e a ound one manganese ca ion bound mainly by he conse ed Asn171 (Asn678, JAK2). The phospha es bind be ween Lys72 (Lys581, JAK2), he Gly- ich loop backbone a Se 53 (Th 555, JAK2), as well as Lys677, JAK2, which pe - o ms he same ole as Lys168 bu is loca ed wo esidues down- s eam along he ca aly ic loop (Figu es 1Band2). Lys677, JAK2 is a lysine o a ginine in all JAK JH2s and has been shown o be essen ial o ATP binding in JAK2 JH2 [45]. This esembles y osine kinases, which use an a ginine a his posi ion o subs i- u e o Lys168 [66]. The hyd ophobic lining o he pu ine-binding pocke is conse ed in all JAK JH2s, including a non-canonically la ge alipha ic esidue in β3 (Leu579, JAK2). JAK2 and JAK1 JH2’s bind ATP wi h mic omola a in- i y [6,45], whe eas he ATP-binding abili y o TYK2 JH2 has been somewha unclea . In hei comp ehensi e TSA sc een o pseudokinases, Mu phy e al. [9], obse ed a sligh posi i e Tm shi upon addi ion o 200 μM ATP o TYK2 JH2, whe eas Toka ski e al. [67] did no obse e signs o ATP binding in hei TSA o compe i ion assays. TYK2 JH2 has, howe e , (along wi h JAK1 JH2) been ound o be able o bind mul iple kinase inhibi o sca olds wi h submic omola a ini y [67,68]. A ecen c ys al s uc u e o adenosine 5-[γ- hio] iphospha e (ATPγS)-bound TYK2 JH2 con i ms ha he domain is indeed capable o bind- ing ATP. Fu he mo e, MANT-ATP, SPR and TSA expe imen s showed ha he domain binds ATP wi h a Kd∼15–20 μM[37]. JAK2 JH2 has been ound o possess low ca aly ic ac i - i y ha phospho yla es wo egula o y esidues (S523, Y570) in JAK2 [6]. Phospho yla ion o hese esidues acili a es he au oinhibi o y in e ac ion be ween JH1 and JH2 [69,70]. JAK1 and TYK2, howe e , do no possess kinase ac i i y, despi e bind- ing ATP [37,65]. Compa ison o he h ee JH2 s uc u es shows ha hey all con ain a helix (αAL) in he ac i a ion loop ha in JAK1 and TYK2, bu no in JAK2, is s abilized by wo sal b idges and may block he subs a e en ance [37]. Fu he mo e, S523 and Y570 esidues a e no conse ed in JAK1 and TYK2 and he lack o sui able subs a es may also accoun o he lack o phospho yla ion-media ed egula ion ha may ep es- en an ances al unc ion in JAK JH2 (JAK2 is he ances al o m). C ys al s uc u es o apo and ATP-bound o ms do no show majo binding-induced changes in TYK2 JH2 [37], and o JAK2 .......................................................................................................................................................................................................................................................................................................................................................................... c 2016 Au ho s. This is an open access a icle published by Po land P ess Limi ed and dis ibu ed unde he C ea i e Commons A ibu ion Licence 3.0. 9 H.M. Hamma ´ en, A.T. Vi anen and O. Sil ennoinen concen a ions a e in he millimola ange [132], so n binding should s ill occu in a physiological milieu e en a ela i ely low a ini ies. Some e y low a ini y ATP binde s (e.g. TRIB2, CASK) ha e e en been hypo hesized o unc ion as cellula n concen a ion senso s [24]. Func ional ATP-binding pocke s a e equi ed o he physiolo- gical unc ion o mul iple pseudokinases, e en in he absence o ca aly ic ac i i y. The de ailed s uc u al consequences o n oc- cupa ion, howe e , a e in many cases no ob ious e en when using mul iple biophysical me hods (e.g. ILK [34]) o when c ys allog aphic s uc u al da a wi h and wi hou n a e a ail- able (see, e.g. JAK2 JH2 [45]). In hese cases me hods able o de ec sub le changes in p o ein dynamics (e.g. NMR o compu- a ional me hods) could p o ide new insigh s. Ne e heless, in mos cases s uc u e-guided, a ge ed, ca e ully con olled mu a- genesis s udies, complemen ed whe e possible wi h a chemical gene ics app oach [133], a e he key o answe ing ques ions o impo ance o n -binding pocke occupa ion. Pseudokinases play an impo an egula o y ole in cellula signalling, and abno mal unc ion o se e al human pseudok- inases has been associa ed wi h human diseases [12]. The mos p ominen examples o his a e p obably he JAK2 JH2 V617F mu a ion unde lying mul iple myelop oli e a i e neoplasms [64], and HER3 o e exp ession in many cance s [134]. In o al, o e 60 mu a ions in he human pseudokinome ha e been shown o cause, o be linked o, a ious malignancies [11–13]. Wi h i s di ec link o pseudokinase egula o y unc ions, he n -binding si e o many pseudokinases poses an in e es ing op ion o pha - macological in e en ions. Fo example, he E351K mu a ion in MLKL inc eases a ini y o ATP and is associa ed wi h hu- man lung ca cinoma [26], and mu a ions in he ATP-binding pocke o JAK JH2 e e he hype ac i a ion o pa hogenic JAK JH2 mu an s [45]. To da e, only a ew pseudokinases, namely HER3 [135], MLKL [136] and TYK2 [67], ha e been a emp- ed o a ge pha macologically, p o iding impo an p oo -o - p inciple esul s ha a ge ing pseudokinases could be a iable he apeu ic op ion. The esul s o pha macological a ge ing o MLKL and TYK2 JH2 a e signi ican and encou aging, bu i should be kep in mind ha he unc ions o pseudokinases a e e y di e se and no as di ec ly p edic able as o ac i e kinases. Fu he de ailed analysis is equi ed o unde s and he physiolo- gical unc ions o di e en pseudokinases, and insigh s hus gained will lay he basis o po en ial u u e he apeu ic a ge ing a emp s. ACKNOWLEDGEMENT We hank J.M. Mu phy o sha ing he TRIB1 s uc u e. 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