AAA+ ATPases: structural insertions under the magnifying glass

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magnifying glass

Matthew Jessop, Jan Felix, Irina Gutsche

To cite this version:

Matthew Jessop, Jan Felix, Irina Gutsche.

AAA+ ATPases:

structural insertions under

the magnifying glass.

Current Opinion in Structural Biology, Elsevier, 2020, 66, pp.119-128.

�10.1016/j.sbi.2020.10.027�. �hal-03071111�

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AAA+

ATPases:

structural

insertions

under

the

magnifying

glass

Matthew

Jessop

1

,

Jan

Felix

2

and

Irina

Gutsche

AAA+ATPasesareadiverseproteinsuperfamilywhichpowera vastnumberofcellularprocesses,fromproteindegradationto genomereplicationandribosomebiogenesis.Thelatest advancesincryo-EMhaveresultedinaspectacularincreasein thenumberandqualityofAAA+ATPasestructures.This abundanceofnewinformationenablescloserexaminationof differenttypesofstructuralinsertionsintotheconservedcore, revealingdiscrepanciesinthecurrentclassificationofAAA+ modulesintoclades.Additionally,combinedwithbiochemical data,ithasallowedrapidprogressinourunderstandingof structure-functionalrelationshipsandprovidedarguments bothinfavourandagainsttheexistenceofaunifyingmolecular mechanismfortheATPaseactivityandactiononsubstrates, stimulatingfurtherintensiveresearch.

Address

InstitutdeBiologieStructurale,Univ.GrenobleAlpes,CEA,CNRS,IBS, 71Avenuedesmartyrs,F-38044Grenoble,France

Correspondingauthors:Jessop,Matthew(matthew.jessop@icr.ac.uk), Gutsche,Irina(irina.gutsche@ibs.fr)

1

Presentaddress:DivisionofStructuralBiology,TheInstituteofCancer Research,LondonSW73RP,UK.

2_Present_address:_Unit_for_Structural_Biology,_VIB_Center_for InflammationResearch,Technologiepark-Zwijnaarde71,9052Ghent, Belgium.

CurrentOpinioninStructuralBiology2021,66:119–128 ThisreviewcomesfromathemedissueonFoldingandbinding EditedbyVicArcusandMargaretCheung

https://doi.org/10.1016/j.sbi.2020.10.027

0959-440X/_ã2020TheAuthors.PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBY-NC-NDlicense( http://creative-commons.org/licenses/by-nc-nd/4.0/).

Introduction:

the

AAA+

domain

architecture

AAA+ ATPases are widely used by cells as motors to powermechanicalworkortoactasmolecularswitchesor scaffolds, often as parts of macromolecular machines [1–4].TheuniversalAAA+ATPasemoduleiscomposed of two subdomains [4–6]. The N-terminal ‘large’ aba subdomain belongs to the ASCE group of P-loop NTPases,and isbuiltaroundacentral5-strand b-sheet carrying the highly conserved Walker A and Walker B motifs, as well as sensor 1 (S1) and arginine finger (R-finger) residues (Figure 1a,b). The Walker A motif (i.e. theP-loop)stabilises ATPbindingwhileWalkerB coordinatesanATP-boundmagnesiumionandprovides

the catalyticglutamate that,together with the polarS1 residue, primes a water molecule for ATP hydrolysis. Oligomerisation of AAA+ ATPases completesthe ATP bindingpocket,with mostAAA+proteins forming ring-shapedhexamers(Figure1c).Oligomerisationallowsthe R-fingertoactintrans,contactingtheg-phosphateofthe ATPmoleculeboundtotheanticlockwiseneighbouring subunit during hydrolysis (Figure 1d). In most AAA+ ATPases, the large subdomain is fused to a ‘small’ C-terminal a-helicallidsubdomain thatcloses over the nucleotide bindingsite andmediatesoligomeric assem-bly.Thissubdomainoftencontributesasecondarginine residue called sensor 2 (S2) to the ATP binding site, whichactseitherincisorintransdependingontheAAA+ ATPase family (Figure 1d). ATPase activity results in relative movements between the large and the small subdomains,whicharepropagatedwithintheoligomeric AAA+assemblyandtransducedtothefunctionaltarget. Thename‘AAA+(ATPasesassociatedwithvarious cel-lularactivities)’highlightstheremarkablediversityofthis protein superfamily, which is due to the fact that the conservedAAA+ATPasemodulecanbeappendedtoa plethoraofdifferentaccessorydomainsconferringavast numberoffunctions[7].Themostcommonfunctionof AAA+proteinsistheATPhydrolysis-driventranslocation of protein or DNA substrates through the central hex-americ channel. Often assisted by additional domains, cofactors, andbinding partners,proteintranslocationby AAA+ ATPasesresultsin target unfolding,disassembly and remodelling, whereas DNA translocation leads to unwinding duringreplicationandtranscription or pack-agingof viralgenomes.

ClassificationofAAA+ATPasesintoclades

In an effort to infer evolutionary relationships and commonfunctionaland mechanisticprinciples between AAA+ATPases,AAA+moduleswereclassifiedintoseven distinctcladesbasedonsequenceandstructural informa-tion available in 2004–2006 [3–5,8]. This analysis revealed that in addition to N-terminal and C-terminal accessory domains, the functions of AAA+ proteins are fine-tunedbyinsertionsofspecificstructuralelementsin the conserved AAA+ core. Each clade is defined as an evolutionary lineageandis furthersubdivided into pro-teinfamilies.Asummaryofthestructuralfeatures defin-ingtheAAA+cladesispresentedin Figure1a,b. Clades1and2aremostlynon-hexamericAAA+proteins and contain DNA polymerase clamp loaders and

Availableonlineatwww.sciencedirect.com

ScienceDirect

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Figure1

(a)

(b)

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Current Opinion in Structural Biology

OverviewoftheAAA+ATPasestructure.(a)Overviewofsecondarystructuralfeaturesandkeysequencemotifs.Thelocationsofclade-specific insertionsintheAAA+coreareindicated(exceptfortheN-terminalandC-terminalhelicesofClade4).(b)3Dstructuresofrepresentative monomersfromeachofClade1–7.Clade1=RFC3(PDBID:1SXJ),Clade2=Orc5(PDBID:5V8F),Clade3=ClpA-NTD(PDBID:6UQE),Clade 4=E1helicase(PDBID:2V9P),Clade5=RuvB(PDBID:1HQC),Clade6=NtrC1(PDBID:4LY6),Clade7=RavA(PDB3NBX).TheClade 6NtrC1structureisenlargedtoshowthelocationofkeymotifsin(a).Keymotifsandinsertionsarecolouredasin(a),aswellastheClade3pore loop1(PL1)coloureddarkgreen.(c)HexamericstructureofLonA(PDBID:6ON2)withmonomersA–Fcolouredindividuallyandboundsubstrate shownasaredsurfaceinthecentreofthehexamericring.Dottedboxesareshownovertwoadjacentmonomers,colouredasin(d).Asideview (below)focussedoncentreofthehexamericringshowaspiralstaircasearrangementofporeloopsaroundthesubstrate.(d)OverviewoftheATP

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DNA-replicativehelicaseloadersrespectively;thefirstis the‘archetypal’ AAA+domain,whereasthesecond has an a-helicalinsertion between b2and a2.Clade3also hasanextraa-helixinsertedbetweenb2anda2,butthis helix is shorter and is followed by a substrate-binding loop(poreloop1orPL1)(Figure1b).Membersofthis ‘classic’ protein-remodelling clade possess a second R-finger and lack an S2 residue. Clade 3 is the most widelystudiedandextensivelyreviewedclade[7],and includesprominentmemberssuchasVps4,katanin,and the N-terminalAAA+domains ofdouble-ringed unfol-dases suchasClpB and Hsp104.Clades4–7allshare a b-hairpin insertion between a3 and b4, before the S1 motif, and are therefore grouped into the pre-sensor 1 insert(PS1i)superclade. Clade4 is composed exclu-sivelyofviralhelicases,andpossessesauniquedomain formed by a-helices N-terminaland C-terminalto the abacoreinsteadofthecanonicalAAA+lidsubdomain. Clade5istermedtheHCLRClade,reflectingthemain familiesthatitiscomposedof—HslU/ClpX, ClpABC-CTD,Lon, andRuvB.MembersofClade5possessno additionalfeaturestothePS1i,andsimilarlytoClade3, areinvolvedinproteinunfoldingandremodelling.Clade 6 ischaracterisedbyab-hairpininsertion ina2termed thehelix-2insert(H2i),andcontainsbacterialenhancer binding proteins(bEBPs)suchasNtrC1andPspF,and the unusual ‘AAA+ GTPase’ McrB. Clade 7 members also possess the H2i, but have an additional a-helical insertion between a5 and a6 called the pre-sensor 2 insert (PS2i). Several functionally divergent families such astheMCM helicase,MoxR,and dyneinfamilies are attributedtothisclade.

Cryo-EMinsightsintotheAAA+ATPasemechanism

As the classification of AAA+ ATPases was performed 15yearsago,onlyaboutadozenhighresolutionstructures of different representatives of this superfamily were available,mostly fromX-raycrystallographicstudies. At this time, the prevailing consensus was that AAA+ ATPases formed symmetric closed rings, with a few isolatedexceptions[3,4,9].Sincethecryo-EMrevolution in2015avastnumberofnewstructuresofAAA+ATPases have been published, and there have been more than 50 publicationspresenting cryo-EMstructures ofAAA+ ATPasessincethestartof2019(SupplementaryTable1). It is now apparent that most AAA+ ATPases show an asymmetric spiral arrangementof six monomers around the central pore (Figure 1c). Many of the published structures wereobtainedinthepresence of asubstrate,

which may play a role in the rearrangement of planar hexamericringsintospiralsto facilitatethreadingofthe substrate through the pore [10–12]. As the binding of ATPisknowntoberequiredforsubstrateengagement, mostofthestructureswereobtainedwithATP binding-efficientbuthydrolysis-defectiveWalkerBmutants,orin the presence of non-hydrolysableATP analogues. Sub-strate-bindingporeloopsformaspiralstaircaseengaging the substrate, with pore loop positions correlating with thenucleotidestate(ATP,ADP,oranapo‘seam’).These structures have allowed the inference of a common mechanism for ATP hydrolysis [7,12] which is based onthesequentialhydrolysisofATParoundthe hexame-ricring,withcorresponding‘hand-over-hand’movements ofporeloopsdrivingunidirectionaltranslocationof sub-stratethroughthepore.However,theuniversalityofthis mechanism is still underdebate [13]. Inaddition, the majorityofstructuresusedtodefinethismechanismare ofproteinsfromtheClassicClade[7,12].Incontrast,the PS1i superclade is much less studied despite its huge functional diversity.In thisreview,we presentan over-view of recent structures of AAA+ ATPases, with a particularfocusonstructuralinsertionsintheAAA+core of PS1isuperclade members.Wediscussthefunctional roles ofthese insertions,and revisit theclassificationof AAA+ ATPases in light of recently solved structures. Finally, we address the controversy surrounding the mechanism of AAA+ ATPaseactivity, underscoring the needforfurtherinvestigationtoclarifythegeneral appli-cabilityof proposedATP hydrolysismechanismsto the entire AAA+superfamily.

Structure

and

function

of

insertions

in

the

AAA

+

core

Clade 3 is by far the best-characterised clade, with many recent high resolution cryo-EM structures of Clade 3 members, including those of the proteasome [14,15–17], Vps4 [18], katanin [19], spastin [20,21], Bcs1 [22], Cdc48/p97/VCP [23–25], and ClpABC-NTD [26–29]yieldingvaluable insightintotheaction of pro-tein-translocatingAAA+ATPases.Asstatedabove,PL1, a short loop before a2, is responsible for binding to substratesduringtranslocation.Theseloopsusually con-tainanaromaticresiduethatnon-specificallyintercalates betweenproteinsubstrateresidues,formingaspiral stair-caseinthecentralpore(Figure1c)[7,12,30].A second-ary loop termedporeloop 2 (PL2), lessconservedthan PL1 [7], islocated betweenb3and a3formsanother spiralstaircasebelowPL1.TherolesofPL1andPL2in StructureandfunctionofAAA+ATPaseinsertionsJessop,FelixandGutsche 121

(Figure1LegendContinued)bindingsiteof(left)Clades1–6(ClpA-CTD,PDBID:6UQE)and(right)Clade7(MCM,PDBID6XTX).Schematicsof twoneighbouringmonomersareshownabove;largeandsmallsubdomainsofoneAAA+monomerarelabelledLandS,whilecorresponding subdomainsfromaneighbouringmonomerarelabelledL0andS0respectively.AsshowninzoomsoftheATPbindingsite(below),the domain-swappedarchitectureofClade7membersmeansthattheS2arginineactsintransandcontactstheg-phosphateoftheneighbouringmonomer, unlikethecanonicalS2ofClades1–6whichactincis.BothbindingsitesareoccupiedbyATPgS,acommonlyusednon-hydrolysableATP analogue.Keymotifsarecolouredasin(a),publicationreferencesforallPDBIDsarepresentedinSupplementaryTable2.

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substratetranslocationareextensivelyreviewedinRefs. [7]and[12].

The Classic Clade isfunctionally similarto theHCLR Clade 5, with members of both being involvedprotein unfolding, remodelling, and proteolysis, as reviewed in Ref.[30].Forsome,suchastheLonprotease,theAAA+ module is fused to a dedicated protease domain. For others,proteasebindingpartnerssuchasClpPandHslV degrade substrates. While Clade 5 lacks the a-helical insertion typical to Clade 3, several Clade 5 members alsointeract withsubstrate viaashort loopbetweenb2 and a2. For other members of Clade 5 as well as both Clade6and7,otherinsertionsintheAAA+core,namely thePS1iandH2i,insteadplayrolesinsubstrate recogni-tionandtranslocation.Despitedifferencesinthelocation oftheseinsertions,thespiralstaircaseconformationseen for the Clade 3 PL1 is conserved across clades,as dis-cussedbelow.

Pre-sensor1insert

ThePS1ib-hairpin,oftenpositionedinthecentreofthe hexamericring,iscrucialtothefunctionofmanyAAA+ proteins in the PS1i superclade. For some, it fulfils a similarroletothePL1inClade3.Intheviralhelicasesof Clade4,thePS1iprotrudesdirectlyintothecentralpore and interacts with double-stranded DNA during origin recognitionand with single-stranded DNA during heli-case unwinding, forming a spiral staircase around sub-strate [31,32]. Similarly, in the Clade 7 DNA helicase MCM, recent structures revealed that the PS1i is involved in coordinating the phosphate backbone of single-stranded DNA during translocation [33,34,

35–37].Forothers,however,thePS1iplaysmorediverse roles.Early studieson RuvB, an evolutionarily distinct memberofClade5,showedthatthePS1iisnotinvolved in substrate translocation but rather in mediating an interaction with its binding partner RuvA [38]. Two recentcryo-EMstructuresoftheClade5unfoldaseClpX boundtosubstratedemonstratethatthePS1iisinvolved insubstraterecognitionratherthantranslocation,withthe majorityofsubstrate-interactingresiduescominginstead from PL1 [39,40]. The structure of the Escherichia coli ClpX reveals that thePS1i reaches into and above the centralporeto engagesubstrate,whichislaterunfolded and translocated through the hexameric pore [40]. In contrast, high-resolution cryo-EM structures of sub-strate-engaged ClpA [26], ClpB [27,28] and Hsp104 [41]haveall beenpublishedrecentlyshowingthatthe PS1iin theC-terminalAAA+ domainisdisplacedaway fromthecentreofthepore(Figure2),withinsertedloops inhelixa2insteadbindingthesubstrateduring translo-cation.Althoughtheseinsertionsareclosetothelocation oftheClade3PL1,theyappeartobemuchlongerandare inthemiddleofa2,bearingacloserresemblancetothe H2iofClade6and7members.Therelativearrangements ofPS1iloopsandthesesubstrate-coordinatingporeloops

fortheClpABC-CTDfamilyaresimilartothoseseenin recentcryo-EMstructuresofLonA[42,43],anotherClade 5 protein, and several members of Clades 6 and 7 (Figure2).

ThePS1iistermedloop2(L2)intheClade6members McrBandbEBPssuchasNtrC1andPspF.InbEBPs,the PS1insertslackthetypicalb-hairpinsecondarystructure andaremoredisorderedcomparedto othermembersof thePS1i superclade, and interact with substrates along withtheH2i(seebelow)[44].Insomecases,suchasfor theMoxRfamilyCbbQ,classifiedintoClade7,thePS1i interactswiththeH2iwhichwouldallowthetransmission ofconformationalchangesfromtheH2itothenucleotide bindingsite[45].

H2insert

SimilarlytothePS1i,theH2ib-hairpinplaysacrucialrole ininteractionswithsubstrateorpartnerproteins.InClade 6bEBPs(extensivelyreviewedinRef.[44]),aconserved motif in theH2i (termed loop 1, L1 or PL1 due to its similarityto theClade 3PL1) facilitatestheinteraction between the AAA+ module and s54-bound RNA poly-merase.bEBPs use theenergy fromATP hydrolysis to remodel RNA polymerase from a closed to an open conformation, thereby activating transcription. The cryo-EM structure of PspF showedthat the H2 inserts of PspF sit in the centre of the hexameric ring and facilitate an interaction with promoter DNA [46]. In NtrC1, anotherbEBP, theH2 inserts are arrangedin a spiralstaircase[47].However,theseproteinsdonotactas motorsbut ratherasmolecularswitches,andassuchare unlikely to translocate substrate through the central channel [44]. The McrB H2i facilitates protein-protein interactionswith theendonuclease McrC, which sits in thecentralhexamericpore,butitisunclearwhetherthe GTPase activity of McrB is used for threading DNA substratethroughthepore [48].

TheH2 insertsof Clade7 members playavarietyof roles.TheH2iofMCMactstogetherwiththePS1ito coordinate substrate DNA, meaning that each MCM protomercontributestwoloops toacontinuous spiral staircase(Figure2)[33,34,35–37].Keyresiduesinthe H2i are necessary for the MoxR protein CbbQ to function as a RuBisCO activase, and in the CbbQ hexamertheH2isprotrudeintothecentralhexameric pore [45]. The H2i also plays important functional roles in the motor protein dynein and the ribosome biogenesis protein Midasin/Rea1. The H2i in the second AAA+ domain of dynein (AAA2) is critical for dynein’s motor activity, with AAA2 H2i mutants still able to hydrolyse ATP but not to perform the power stroke associated with motor function [49]. Cryo-EM structures of Midasin/Rea1 showed that AAA2H2iisinsteadextendedbyana-helicalbundle, whichsitsinthecentreofthehexamericringasaplug CurrentOpinioninStructuralBiology2021,66:119–128 www.sciencedirect.com

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and inhibitsATPaseaction ofthehexamer whennot boundtoitssubstrate[50,51].Additionally,therecent cryo-EMstructureofRea1incomplexwith theRix1/ pre-60S ribosome particle showed that the H2i of AAA2 promotes an interaction with Rix1 while the

H2i of AAA5 instead contacts the Rea1 MIDAS domain [52]. However, it is unclear whether these pore loop-facilitated interactions are involved in threading substrate, or rather in mediating protein-protein interactions alone.

StructureandfunctionofAAA+ATPaseinsertionsJessop,FelixandGutsche 123

Figure2

HexamerstructuresofselectedPSI-insertsuperclademembersshowingPS1i,H2iandPS2ilocations.Colouredboxesindicateclade

classification:red=Clade4,magenta=Clade5,green=Clade6,blue=Clade7.InsertionsarecolouredasforFigure1(largeAAA+subdomain =lightbrown,smallAAA+subdomain=orange,PS1i=magenta,H2i=green,PS2i=navyblue,Clade4-specificN-terminalandC-terminalhelices =lightblueandcoralrespectively,grey=inter-protomerlinkers).Boundsubstrates,whenpresent,arecolouredred.AllPDBIDsarelistedin

Figure3d,exceptforLonA(6ON2),MCM(6XTX)andRavA(6SZB),andpublicationreferencesforallPDBIDsarepresentedinSupplementary Table2.

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Figure3

(a)

(b)

(c)

DiscrepanciesinAAA+ATPaseclassification.(a)ComparisonofmonomerstructuresforClade7members.WhileMCM,RavA,BchIand CHU_0153(notshown)possesstheclade-definingPS2i(colourednavyblue)anddisplayrepositionedsmallAAA+subdomains,CbbQ,Dynein (AAA2monomershown)andRea1(AAA3monomershown)insteaddisplayacanonicalarrangementofAAA+subdomains.(b)Comparisonof selectedClade5and6monomers,focussedontheregionsurroundinga2(lightblue)anda3(mediumblue).Insertionsina2arecolouredlight greenandthePS1iiscolouredmagenta.WhiletheClade5membersClpX,RuvB,TorsinandHslUpossessacontinuousa2,ClpA-CTD, ClpB-CTD,LonAandLonBpossessaninsertionverysimilartothoseseenforClade6membersNtrC1,PspF,FlrC,McrB,aswellasFleQandZraR (notshownbutalmostidenticaltoNtrC1).ClpC-CTDandHsp104-CTDstructures(3PXIand6D00respectively)lackbuilt-inresiduesatthis location,howeverthenumberofmissingresiduesatthebreakina2areconsistentwithaninsertedloop.(c)Structuralsimilaritydendrogramof

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Pre-sensor2insert

ThePS2i,thedefiningfeatureofClade7AAA+ATPases, formsalonga-helixthatdrasticallyrepositionsthesmall subdomain relativeto thelarge subdomain[4].Despite this repositioning, the overall arrangement of large and smallsubdomainsinthehexamerandthearchitectureof theATPbindingsite ispreserved(Figure 1d).In other AAA+ clades, the linker between large and small sub-domainsoperatesasahinge,withATPhydrolysislinked toconformationalchangeswithinamonomer.Therecent cryo-EMstructureoftheClade7ATPaseRavAfromthe MoxR familyshowed that thesehinge-likemotions are conserved,butoccurinsteadbetweenthelargeandsmall subdomainsofneighbouringmonomers[53].Inaddition, similaritiesbetweentwofoldsymmetricclosedringstates of ClpX[54]andRavA[53],andin particularthe pres-enceofanucleotide-free‘doubleseam’,supporttheidea ofaconservedATPasemechanismbetweenClades5and 7, despite thehuge differences in domain architecture. Althoughthereareseveralrecentcryo-EMstructuresof Clade 7ATPaseswiththePS2i,inparticularMCMand RavA [34,35–37,53], the effects of AAA+ subdomain repositioningarenotwell-explored.Clade7ismuchless characterisedthanotherclades,andfurtherinvestigation may uncover the role of the PS2i in modulating the ATPaseactivity.Finally,severalAAA+proteinsclassified into Clade 7 clearly lack the PS2i, as discussed below, raisingquestionsaboutthecurrentclassificationscheme.

Discrepancies

in

AAA+

classification

Just as thevast numberof recent AAA+ ATPase struc-tureshasfacilitatedtheincreasedunderstandingoftheir molecularmechanism,italsooffersusachancetomake extensive structuralcomparisons and reveals discrepan-cies in thecurrent classification scheme, particularlyin thePS1isuperclade.Becausethegroupingintocladesis often used to extend hypotheses from one protein to membersofthesamecladeandtocontrastobservations between members of different clades [4,42], working within the framework of an accurate and up-to-date classification system seems important, in particular for theinvestigationofthegeneralapplicabilityofcurrently proposed AAA+ ATPase mechanisms, as discussed below.

Members of Clade 7 offer the most apparent example of discrepancies in classification. Indeed, structural alignment of Clade 7 AAA+ ATPasesshows that while severalmemberspossessthecharacteristicPS2i,dynein, Midasin/Rea1andCbbQlackitandretainthecanonical

arrangement of large and small AAA+ subdomains (Figure 3a) [53,55]. In addition, although dynein is classified an H2i AAA+ ATPase, the sequence stretch originallyidentified[3]asbeinganH2iinAAA3,thethird tandemAAA+domain,infactappearsasaloopbetween a2and b3whiletheonlyinsertionin themiddleof a2 appearstobeinAAA2[55].Thissuggeststhateitherthe other five AAA+ domains in dynein have subsequently lost theirH2i,orprobablymorelikely,therewasalater independent insertion intoa2of AAA2 afterbranching fromotherPS1iproteins[55].Incontrast,allsixtandem AAA+ domainsinMidasin/Rea1possess anH2i[50]. Asintroducedabove,severalmembersofClade5,namely ClpA-CTD,ClpB-CTD,ClpC-CTD,Hsp104-CTDand Lon also have insertions in a2 that interact with sub-stratesinasimilarwaytotheH2isofClade6and7.On thefaceofit,thisfeaturewouldplace theseproteinsin theH2i-containingClade6.Comparisonofthemonomer structures of these proteins with those of Clade 6 and 7 shows a high degree of structural conservation (Figure 3b). However, as is possibly the case for the H2iintheAAA2domainofdynein,itisconceivablethat these insertions arose independently multiple times. Indeed,astructuralsimilaritydendrogrambasedon struc-turalalignmentofmonomersacrossthePS1isuperclade, generated using the distance matrix-based structural alignment algorithmontheDALI server[56],does not maintain asingle grouping for proteins containingH2is but rather splits them into several different groups (Figure 3c). Therefore, the original classification of AAA+ ATPasesbased onstructural PS1i,H2i andPS2i insertionsandassumingtheiremergenceonlyonceinthe course of the superfamily evolution may be outdated. The evolutionary historyof theAAA+ superfamilymay be more complex, suggesting that a fresh look at the classification system based on large-scale analysis is needed in light of the ever-growing structural informa-tion, particularly for the comparatively understudied Clades 6 and 7. In addition, some proteins such as Pch2/TRIP13 do not fit in the current classification scheme, but possess features of multiple clades [57]. Finally,itmaybeworth askingwhetherisitstill useful to referto clades atall,or whether itis betterto group AAA+ATPasesaccordingtotheirfunctionalsimilarities.

Perspectives

—

a

universal

AAA+

ATPase

mechanism?

Although the recent wealth of structural information has yielded substantial insight into the mechanism of StructureandfunctionofAAA+ATPaseinsertionsJessop,FelixandGutsche 125

(Figure3LegendContinued)PSI-insertsupercladeAAA+ATPasesgeneratedfromastructuralalignmentusingDALI[56]withoutsidelines colouredaccordingtothecladeclassification.The‘all-against-all’optionwasused—thiscarriesoutsequentialpairwisecomparisonsbetweenall PDBfiles,generatingdistancematricesthatdescribedistancesbetweenequivalentCaatoms,andhierarchicallyclustersthesematricesintoa Newickformatdendrogram[61].Wherenecessary,missingloopresiduesweremodelledusingthePhyre2server[62]beforestructuralalignment. DendrogramvisualisationwascarriedoutusingiTOL[63].AtableofPDBIDsusedasinputforthecreatingthedendrogramisshownontheright, withproteinscolouredbyclade.PublicationreferencesforallPDBIDsusedarepresentedinSupplementaryTable2.

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AAA+ATPaseactionandtheorthodox‘hand-over-hand’ modelofsequentialATPhydrolysishasbecomewidely accepted,severalrecentpublicationscallitintoquestion [30,40,53,58]. For example, high-speed atomic force microscopyofthehistonechaperoneAbo1hasprovided directevidenceforATPhydrolysisatnon-adjacentsites inthehexamericring[58].Inparallel,tworecent cryo-EMpapers ontheClade 5 unfoldase ClpXP have reig-niteddebateoverwhetherasequentialorstochasticATP hydrolysis mechanism is correct by interpreting very similar structures in terms of two very different but plausible mechanisms [13,39,40]. Despite the attrac-tiveness of the idea of a unifying ATPase mechanism forallAAA+proteins,therealitymaybemorenuanced.It isconceivablethatdependingoncellularconditionsand interactions with binding partners or cofactors, AAA+ ATPasesmaybeabletoswitchbetweenstrictly sequen-tialand stochasticmodesof action [7,30].Inaddition, whilemostAAA+ATPasesactasmotorswithcontinual ATPturnover,otherssuchastheClade1clamploaders, Clade2helicaseloadersandClade6bEBPsinsteadactas ‘switches’,withasingleATPasecyclelinkedtoasingle event. Whether these switch-like ATPases all function withthesamemechanismasthemore extensively char-acterisedAAA+ ATPases‘motors’isstill uncertain[44], although the recent cryo-EM structure of the Clade 1RFCsuggeststhatthisisunlikely[59].Besides,other non-conventional AAA+ proteins exert force on their protein substrate laterally such as dynein, or act on membranes via unexplored mechanisms such as the unusualClade5ATPaseTorsin,whichformslonghelical polymers[60].Therefore,inparalleltoallowing investi-gationofmechanisticcommonalities,thecurrentflurryof cryo-EMstructuresmayalsoinspirethedisentanglingof cladeorevenprotein-specificfunctionaldifferencesand theirstructuralunderpinning.

Conflict

of

interest

statement

Nothingdeclared.

Appendix

A. Supplementary

data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10. 1016/j.sbi.2020.10.027.

Acknowledgements

WewouldliketothankourcolleaguesfromtheIBSMICAgroupfortheir constantsupportandinterestinourworkonAAA+ATPasesandthe stimulatingscientificatmosphere.ThisworkwasfundedbytheEuropean Union’sHorizon2020researchandinnovationprogrammeundergrant agreementNo.647784toIG.MJwasaCEA-fundedPhDstudent.JFwas supportedbyalong-termEMBOfellowship(ALTF441-2017)andaMarie Skłodowska-CurieactionsIndividualFellowship(789385,RespViRALI).

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