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Slicing a : Structural features of the ATP-dependent Lon gleaned from investigations of isolated domains

Tatyana V. Rotanova, Istvan Botos, Edward E. Melnikov, Fatima Rasulova, Alla Gustchina, Michael R. Maurizi and Alexander Wlodawer

Protein Sci. 2006 15: 1815-1828 Access the most recent version at doi:10.1110/ps.052069306

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REVIEW Slicing aprotease: Structural features of the ATP-dependent Lon proteases gleaned from investigations of isolated domains

TATYANAV.ROTANOVA, 1 ISTVAN BOTOS, 2,3 EDWARD E. MELNIKOV, 1 FATIMA RASULOVA, 4,5 ALLA GUSTCHINA,2 MICHAEL R. MAURIZI,4 2 AND ALEXANDER WLODAWER 1 Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry,Russian Academy of Sciences, Moscow117997, Russia 2 Macromolecular Crystallography Laboratory,National Cancer Institute at Frederick, Frederick, Maryland 21702, USA 3 Laboratory of Molecular Biology,National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, USA 4 Laboratory of Cell Biology,National Cancer Institute, Bethesda, Maryland 20892, USA 5 Enteric Diseases Department, Infectious Diseases Directorate, Naval Medical Research Center,SilverSpring, Maryland 20910, USA

Abstract ATP-dependentLon proteasesare multi-domain enzymesfound in alllivingorganisms.All Lonproteases containanATPasedomainbelonging to theAAA+ superfamilyofmolecularmachinesand aproteolytic domain with aserine-lysine catalyticdyad. Lonproteases canbedivided into twosubfamilies,LonAand LonB,exemplified by the Escherichiacoli and Archaeoglobusfulgidus paralogs,respectively. TheLonA subfamily is definedbythe presence of alarge N-terminal domain,wh ereasthe LonB subfamilyhas no such domain,but hasamembrane-spanningdomainthatanchors theprot eintothe cytoplasmicsideofthe membrane.The twosubfamilies also differ in theirconsensus sequences. Recent crystalstructuresfor several individual domainsand sub-fragmentsofLon proteaseshavebegun to illuminate similarities anddifferences in structure–function relationshipsbetween thetwo subfamilies. Differencesinorientation of theactivesite residues in severalisolatedLon protease domainspoint to possible rolesfor theAAA+ domainsand/or substrates in positioningthe catalyticresidueswithin theactivesite. Structures of theproteolytic domains have also indicatedapossible hexamericarrangement of subunits in thenativestate of bacterialLon proteases. Thestructure of alarge segmentofthe N-terminal domain hasrevealedafoldingmotif present in otherprotein families of unknownfunctionand should lead to newinsightsregarding ways in whichLon interactswith substrates or othercellularfactors.These firstglimpsesofthe structureofLon areheraldingan exciting newera of research on this ancientfamilyofproteases. Keywords: AAA + protein; Lon protease; ATP-dependent protease; Ser-Lys dyad; LonA and LonB subfamilies; domains; crystal structure

The Lon family of peptidases Reprintrequeststo: Alexander Wlodawer,Macromolecular Crystal- lography Laboratory,NCI at Frederick, P.O. BoxB,Frederick,MD The Lonprotease family (MEROPS [Rawlings et al. 21702, USA; e-mail:[email protected]; fax: (301) 846-6322;or TatyanaV.Rotanova,Shemyakin–OvchinnikovInstitute of Bioorganic 2004] clan SJ,family S16),which is conserved in the Chemistry,Russian Academy of Sciences,Miklukho-Maklayast. 16/10, andineukaryoticorganelles such as mito- GSP-7, Moscow 117997, Russia; e-mail: rotanova@.siobc.ras.ru; fax: +7-495-3357103; or Michael R. Maurizi, LaboratoryofCell chondria and peroxisomes, is themostwidespread family Biology,NationalCancer Institute, Bethesda, MD 20892, USA; e-mail: of ATP-dependent proteases. Prokaryotic Lons are key [email protected];fax:(301) 480-2284. Article and publication are at http://www.proteinscience.org/cgi/doi/ responsible for intracellularproteolysis, con- 10.1110/ps.052069306. tributing to protein quality and cellular homeostasis by

Protein Science (2006), 15:1815–1828. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2006 The Protein Society 1815

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eliminating mutant andabnormalproteins andparticipat- Lons are dividedinto twosubfamilies, LonA and inginrapid turnoverofselect short-livedregulatory LonB,based on differencesinthe number of domains proteins(Goldberg1992;Gottesman and Maurizi 1992; and characteristic sequences within the domains (Rota- Gottesman 1996;Wickner et al.1999). Though less well nova et al.2003, 2004). Both subfamilies contain the studied,eukaryotic Lons have been shown to exhibit ATPase (A) domains that include typical AAA + modules, similarly important regulatory and protein quality control as well as theproteolytic (P) domains, butwhereas LonA functions in mitochondria (Van Dycket al.1994; vanDijl enzymes contain alarge N-terminal(N) domain, the et al. 1998;Botaand Davies 2002). Lon,like allother LonB enzymeshavenoN-domain buthavealarge ATP-dependent proteases (FtsH, ClpAP,ClpXP,HslVU, transmembrane domain insertion within the AAA + mod- andthe 26 Sproteasome), belongstothe AAA+ protein ule betweenthe Walker motifs Aand B(Fig. 1).Ithad superfamily (ATPases associated with diverse cellular been suggestedthat Lons are proteases(Chungand activities) (Neuwald et al. 1999;Maurizi and Li 2001; Goldberg1981;Waxman andGoldberg1982; Goldberg Oguraand Wilkinson 2001; Lupas andMartin 2002;Iyer et al. 1994), althoughthe amino acid sequencesofLonA et al. 2004). In additiontoprotein unfoldingand pro- andLonBP-domainsshownohomologywith serine teolysis, AAA + proteinsare involved in many cellular proteasescontaining the classicalcatalytictriad Ser- processes, including membrane fusion, protein andor- His-Asp(Amerik et al.1988, 1990,1991; Goldberg et al. ganelle translocation, DNAand RNAunwinding, assem- 1994; Rotanovaetal. 2004), or,indeed, to any other bly anddisassembly of multi-protein complexes, and proteases. Active-site-directed inhibitors of classical serine microtubulesevering,toname thebestknown. The proteases, such as sulfonyl fluorides, chloromethyl ketones, cellular activity of AAA+ proteins is largely definedby or fluorophosphates, are poor inhibitors of Lon, and none the functional partners with which theyassociate. In the has been shown to modify the residues. Com- case of Lon, theAAA+ domainiscovalently fusedto parative analysisofthe primary structuresofthe Lonpool aproteasedomain. and site-directed mutagenesis of the full-length Escherichia Lonproteases function as oligomeric assemblies, coli Lon(EcLon) (Rotanovaetal. 2003, 2004), along with whichhad been variouslyreportedtoconsist of four to the recently determined structure of the EcLonP-domain eight identical subunits (Goldbergetal. 1994;Roudiak (Botosetal. 2004b), established that the activesites of Lon et al. 1998; Lee et al.2004). Anumber of direct and proteases haveaSer-Lys catalytic dyad (Ser679 and Lys722 indirect observations indicate that bacterial Lons form in the EcLon numbering). ringsconsisting of six subunits(Leeetal. 2004; Park Thedomainsin EcLon,arepresentative member of the et al. 2006). The yeast mitochondrial Lon has been shown LonA subfamily,are assigned by us for thepurpose of to form seven-membered rings(Stahlbergetal. 1999), and this review as 1–309(N-domain), 310–584 (A-domain), similararrangementsmight also be presentinproteinsfrom and 585–784 (P-domain), althoughthere is no consensus othersources.The subunits themselves arecomposedof on such adivision at this time andthe exactposition threeormoreindependently folded domains(seebelow). of domain boundaries, especiallybetween the N- and

Figure1. Schematic diagram of the typical members of LonA and LonB subfamilies, represented by E. coli and A. fulgidus Lon proteases, respectively.Domain definitions are explained in the text.(Dark blue) The segments for which three-dimensional structures have been solved; (green) the locations of the Walker Aand Bmotifs (AAA+ module), marked Aand B; (red and orange) residues forming acatalytic dyad (serine [S] and lysine [K]); (yellow) the transmembrane domain of the LonB proteases; (magenta) the sensor-1 and sensor-2 regions. The positions of putative intein insertions located just after the TM domains in some LonB proteases (but not in AfLonB) are not shown.

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A-domains, may be subject to future revision. AAA+ with different substrates,reflecting size, global andlocal modules generally contributetotargetselection and thermodynamicstability,and interactions of unfolded regulation of the activity of the associated functional substrates with various binding sites along the trans- component(theproteasedomain in the case of Lon) location pathway in the AAA+ module andthe protease (Neuwald et al. 1999; Wickner et al.1999; Ogura and component. Wilkinson 2001; Lupas andMartin 2002; Iyer et al. Understanding themechanism andregulation of the 2004). Basic AAA+ modules consist of two domains: activities of these complex enzymes will require exten- alargernucleotide-binding domain ( a / b -domain) and sive structuralinformation on thefunctional domains and asmaller helical domain ( a -domain). The a / b -domain theinterfaceand interactions between them. Structural containsconserved motifs, including Walker Aand Band detailswillvaryfor different families because of varia- sensor-1, whichtake part in nucleotidebindingand tions of the mode of theinitial binding interactions hydrolysis, andan‘‘Arg finger’’ that contributes to between substrates andATP-dependentproteases, the activation of ATPhydrolysis upon subunit interaction need for effective force generation without disruption (Neuwald et al. 1999). The a -domain contains at least of the enzyme itself, the complexity of translocating oneconserved motif, sensor-2,containing an Arg(or aprotein through thenarrowaxial substrate channels, rarely Lys) residue, also involved in ATPhydrolysis and andallosteric effects leadingtoactivationofthe pro- in protein substrate remodeling (Neuwald et al.1999; teolytic site or conformational changes needed to release Iyeretal. 2004). reactionproducts. So far, significant progress in structure The bindingand hydrolysis of ATPpromotes acycle of determinationhas been made with ATP-dependent conformational changes within the AAA + protein,and proteasesthatare assembledfromindependently ex- relative movement between the a / b -and a -domains during pressed AAA+ proteinsand protease components,such the nucleotide binding and hydrolysis cycle generates a as the Clp proteases (Wangetal. 1997; Guoetal. 2002; mechanical force that acts on the functional partners and Kim andKim 2003)and proteasome-relatedproteases associated substrates (Ogura and Wilkinson 2001; Iyer et al. (Lowe et al.1995; Bochtler et al. 1997,2000; Groll et al. 2004). Thus, many AAA+ proteins function as chemo- 1997; Sousa et al.2000; Sousa andMcKay 2001;Wang mechanical enzymes to alter the conformation of proteins, et al. 2001). Thesestructures provide keyinsights into disassemble macromolecular complexes, or translocate functionally important featureslikely to be shared by all macromolecules between different compartments within such enzymes. TheATP-dependentproteases, Lon and the cell (Baumeister et al. 1998; Langer 2000; Lupas and FtsH, in which theAAA+ protein andthe protease are Martin 2002; Gottesman 2003; Sauer et al. 2004; Wang fused in asingle polypeptide together with one or more 2004; Groll et al. 2005; Martin et al. 2005). other functional domains, present aunique challenge. The ATPase components of several ATP-dependent Although their global architectural features, such as the proteases have been showntohaveprotein unfolding fold of theAAA module, areexpectedtobeconserved,as activity or at least the ability to locally disrupt the has been already shown forFtsH (Krzywda et al. 2002; structureofatargetprotein (Weber-Ban et al.1999; Niwa et al. 2002), thesingle-polypeptideATP-dependent Kimetal. 2000;Singh et al. 2000). Foramajority of proteases mightdifferinimportant mechanistic and ATP-dependent proteases, structuraldestabilization is a structural details related to changesinthe interface be- prerequisite for allowing substrate proteins access to the tweenthe A- andP-domains and allosteric communi- proteolytic active sites, whichare sequestered in internal cation betweenthem. In fact,the structural analysis of chambers within the holoenzyme complexinsuch away Lons has already provided afew surprises andhas ledto that bound substrates must passthroughanarrowaxial areconsideration of howthis classofATP-dependent channel to reachthem (Hegerletal. 1991; Lowe et al. proteases works. Forexample, thestructure of the 1995;Bochtler et al.1997; Wang et al. 1997; Groll et al. isolated protease domain (Botos et al. 2004b)does not 2000). Once protein substrates enter the proteolyticsites, showthe kind of secluded degradation chamber observed peptidebond cleavage occurs rapidly andpromiscuously in the proteasomes and Clpproteases, suggesting that andwithout anyfurtherexpenditure of energy,giving protectionofcellularproteins from degradation might rise to avariety of peptide products ranging in size from rely on of the catalytic activity of the threeto ; 15 residues(Kisselevetal. 1999;Choiand proteaserather than blocking access to thesesites by Licht2005). The translocation process itself is also compartmentalizingthem. energetically unfavorable, andATP hydrolysisisneeded Crystallization of intactLon proteases hasbeen to drive this process (Hoskinsetal. 1998;Singhetal. attempted formanyyears withoutsuccess. Fortunately, 2000;Burton et al.2001; Reid et al.2001; Kenniston limited identified anumber of stable domains et al.2003).The distributionofthe energy requirement andtheir combinations (Ovchinnikovaetal. 1998;Roudiak between unfolding andtranslocationislikely to vary and Shrader 1998; Vasilyeva et al. 2002; Patterson et al.

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2004; Rotanovaetal. 2004), which has allowed successful purification and crystallization of several Lon fragments, as well as their cloned and expressed equivalents. In some cases, both approaches were used in the study of the same domain, providing complementary information (Botos et al. 2004b, 2005). At this time, detailed structuralinformation is available for asubdomain of the N-domain and for the a -domain of EcLonA, as well as for the P-domains of wild-type and mutated forms of EcLonA, Archaeoglobus fulgidus LonB ( AfLonB), and Methanococcus jannaschii LonB ( MjLonB). The available structuresprovide an in- complete picture of Lon molecules and their oligomeric complexes, but they havebeen valuable in defining struc- tural constraints on the domain organization and assembly of the native protein and in providing information explain- ing the mode of catalytic activity.

The N-terminal domain of LonA The N-domain is found only in LonA subfamily mem- bers. Intriguingly,the LonN-domain is related to a widespread anddiverse family of proteins of unknown biological function that arepresent as 200–300 amino acid–long open readingframes in thegenomes of avariety of organisms (Lietal. 2005). The LonN-domain is composed of twoormore smaller domains identified by acombination of multiple sequence alignment andlim- ited proteolysis. Limited proteolysis of EcLonA produced several transiently stable N-terminal fragments terminat- ing between Lys223and Lys239(cleavage with or lysylendopeptidase C), after Glu240 (Staphylococcal V8 protease), and betweenTyr228and Met234and after Trp303 () (Ovchinnikova et al. 1998; Vasilyevaetal. 2002;Patterson et al.2004; T.V. Rotanova, I. Botos, E.E. Melnikov, G.G. Leffers, F. Rasulova, A. Figure2. Crystal structures of the fragments of E. coli Lon. ( A )Structure Gustchina, M.R. Maurizi, andA.Wlodawer, unpubl.).Upon of the N-terminal subdomain of the N-domain (designated EcLon-N119, extended incubation with chymotrypsin, the N-terminal PDB code 2ane), shown as aribbon diagram in rainbowcolors (changing domain wasreduced to one very stable fragment, Lon-N209, from blue at the Nterminus to red at the Cterminus). ( B )Structure of the a -domain from the AAA+ module, shown as aribbon diagram in rainbow which exists as amonomer in solution and is structurally colors (PDB code 1qzm). The side chain of sensor-2 Arg542 is marked in homogeneous, judging from its ability to yield large stick representation. ( C )Structure of the P-domain, shown as aribbon crystals.The occurrence of insertions within divergentLon diagram in rainbowcolors (PDB code 1rr9). The side chains of the sequences suggestedanother possible boundaryinthe catalytic dyad are marked in stick representation. region near EcLonA residue 119; aconstruct of EcLon- N119 was expressed and purified, and its structure solved (Li et al. 2005). EcLon-N119 has anovel fold made up of BPP1347, whosefunction and level of expression (if any) three twisted b -sheets folded into ashallowUshape with are not known, is the product of a202-amino-acid open asingle a -helix nestledinthe depression (Fig. 2A).Most reading frame randomly isolated and crystallized as part of the surface,including the region aroundthe a -helix,is of astructural genomics effort (PDB code 1ZBO).The hydrophilic, with the exception of abroad swath of exposed BPP1347 molecule has two domains connected in tandem hydrophobic residues cutting across the b -sheets on the by asingle linker polypeptide segment. Its N-terminal surface opposite the a -helix. domain has 19%sequence identity to Lon-N119, and, re- Astructure similar to that of Lon-N119 wasalso markably,asimilar degree of identity is alsoseen through- observed forthe b -rich N-terminal domain of asmall outthe C-terminal part of BPP1347 andresidues 120– protein, BPP1347, purified from Bordetella parapertussis. 209of EcLonA. The C-terminal domain of BPP1347

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containsfour prominent a -helices with unique topology, The a -domain of the AAA + module and this structure correlates very well with the secondary The AAA + module of Londid not yield crystals suit- structure predicted for residues120–209 of Lon(EcLonA able forstructuredetermination; however, of numbering) based on the sequences of >100 LonA proteins, EcLonA by a -chymotrypsinyielded astablefragment including the number and length of the helicesand the consistingofresidues 491–584, which was purifiedand positions of turns and loops. We postulated that the ; 210 crystallized (Botos et al.2004a). This largely a -helical amino acid–long N-terminal fragments of EcLonA and fragment represents the complete small a -domain of the otherprokaryotic A-typeLon proteins have thesame AAA + module anddisplays aconserved topology (helix- overall structure as BBP1347(Li et al. 2005). In strand-helix-helix-strand-helix) reported formanysimilar BPP1347, thereisalarge interface between the two a -domains (Lupas andMartin 2002) (Fig. 2B). Helix 1is subdomains formed mostly by hydrophobic andhydrogen bonding interactions.The structure of Lon-N209 mod- slightly bent in the middle. The significance of thebend is eled on BPP1347also shows an extensive interface unclearatpresent, although it alters the space and the angle between the a -domain andthe larger a / b -domain. involving >20side chain interactions, suggesting that + thetwo subdomainsofLon N-domains are fixedrelative AAA proteinsdifferdepending on whether helix1is to each other and function as asingle bimodal structural straight,isbentinthe middle, or contains atwo-amino- unit. acid bulge in themiddle(Ogura andWilkinson 2001). The structural similarity between BPP1347 and frag- Following b -strand 1and helix 2isalong helix 3, at the ments of theN-domain of Lonsuggests that thetwo might beginning of which is located thewell-conserved sensor-2 have some activity or property in common. It is not residue, Arg542.The following b -strand loops form knownif, andunder what conditions, BBP1347orother aparallel b -sheet with the b -strand 1. TheC-terminal hypothetical proteinsconsisting only of this conserved helix 4isunwound at theend where it should be con- domain and encodedinthe of manyother organ- nected to thebeginning of theP-domain. Current models of thefunctionofAAA+ modules of isms are expressed in vivo.The N-domain of EcLonA + appears to have some protein-binding ability and might typical AAA proteins suggest that the a -domain acts as contribute to substrate recognition. E. coli Lon lacking arigid bodyconnected to the a / b -domainbyaloopthat 107N-terminal residueshad drastically reduced protein- is sensitive to thenucleotide state of the module (Rouiller degrading activity in vitro (Rasulova et al. 1998a). et al. 2002; Wang 2004; DeLaBarre andBrunger 2005). Mycobacteriumsmegmatis Lonlacking90, 225, or 277 Stability of subunit interactionsinthe assembled rings N-terminal residues lost practically all proteolytic activ- is conferred by theinteractionofthe a -domain of one ity while exhibiting reduced protein binding activity,as subunit with the a / b -domain of an adjacent subunit well as alteration of the oligomeric state (Roudiakand (Lenzen et al.1998; Bochtler et al.2000;Zhang et al. Shrader 1998). It is possible,however, that such deletions 2000).Binding andreleaseofanucleotide causearelative can cause structural perturbations that affect activity in rotation andseparation betweenthe a -domain andthe otherparts of the proteinand influence itsoligomeriza- a / b -domain (Wang2004). Depending on thestate of tion (Lee et al.2004). Amore specific indication of assembly and thepresence of aboundload, this relative substrate interaction by the N-domain is the identification movement can impose aforceonthe load, impose aforce of an E. coli Lonmutant altered in substrate specificity on apolypeptideextension connected to one of the (Ebel et al.1999). Amutation in Lonthat converts domains, or cause partial separation of thesubunits. In Glu240 to Lysresultsinstabilization of oneLon sub- thefixed-ring configuration, movement between the strate,RcsA, in vivo butdoes not affect the degradation of a -and a / b -domainswould forcethe a / b -domaintobe anothersubstrate,SulA.Whether Lonhas specific protein displaced along the axis andexert aforce on asubstrate recognition sites in theN-domainremains to be proven. It polypeptidewithin the axial channel. If motion of the is possible thatthe N-domaincontributes to specific a / b -domain is restricted, movementbetween the a -and substrate interactionbybinding disordered regionsin a / b -domainswill cause the a -domain to rotate out andup substrates that also carryspecific motifs recognized else- from the ring (Wang2004), and, in the case of Lon, this where on Lon. There is evidence of such an auxiliary role effect will impinge on the secondary structural elements forthe N-domain of ClpA, thechaperonecomponent of by which theP-domain is connected to the AAA + module. ATP-dependentprotease, ClpAP (Xia et al.2004). We In this way,nucleotide binding or release canallosteri- speculate that themoredistantly related domainsand cally affect interactions between thestructuralelements stand-aloneproteins that arestructurally similar to the of the P-domain or eventhe configuration of thecatalytic LonN-domain mightalso bind unfolded proteins or residues at theactive site.Taking into account the disorderedpolypeptides.Suchactivity shouldprovide oligomeric structureofLon andassuming thepresence some clue as to their stillunknown functions. of multiple active subunitsinthe oligomer,itislikely that

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thesedivergenteffects occurondifferent subunitsinan ordered or sequential manner, similarlytoATP syn thase subunitsthat are sequentially active,depending on rotary interactions with the g subunit (Stock et al.2000).

The structureofthe P-domain of Lon In vitro, purified EcLon P-domain exhibits no detectable activity against protein substratesdegradedbyfull-length Lon(Botos et al.2004b), butretainsasignificantfraction of peptidase activity (Rasulova et al.1998b). Interest- ingly,aconstruct containingresidues 793–1133ofyeast Lon,which comprises theP-domain along with most of the a -domain,exhibited lowbut significant proteolytic activity in vivo (van Dijl et al. 1998). Moreover, activity of this yeast Lon a P-fragmentwas enhancedwhenitwas coexpressedwith aconstruct containing theN-and A-domains(residues 1–917), indicating that theP-domain is somewhat malleableand mayrequire interactionswith other parts of Lon to maintain afully active configuration (van Dijl et al. 1998). Thecrystalstructureofthe P-domain of EcLonA (residues585–784, Fig. 2C) (Botos et al.2004b) eluci- dated auniquefold that is shared only with the sub- sequently determined P-domains of MjLonB(residues 456–649) (Im et al.2004) and AfLonB (residues 417–621) (Botos et al. 2005), as wellaswith viral protease VP4 (Feldman et al. 2006); this structure will be detailed below, with only thedifferences outlined for theother two Figure3. Comparison of the structures of the P-domains of E. coli LonA structures(Fig. 3). (green), A. fulgidus LonB (cyan), and M. jannaschii LonB (magenta). ( A ) The first crystals of the P-domain of EcLonbelongedto Ribbon diagram of the superposition of the main chains, with the side chains in the active site marked in sticks. ( B )Detailed superposition in the aP3 1 spacegroup. Theasymmetric unit containedsix vicinity of the active site, with the trace of the chain that contains the subunits, and the putative oligomeric molecule appeared catalytic serine shown as aribbon. The position of the hydroxyl of Ser679 as aringwithpseudo-sixfold molecular symmetry.The in EcLonA is modeled,whereas all other atoms are from experimental P-domain subunititselfhas six a -helices andten b -strands structures. (Fig.2C) and is composed of two compact subdomains, residues585–697 and 698–784. The first nine residuesof the P-domain (585–593) are disordered. The b -strand1 b -loopformedbyantiparallel strands7and 8, followed and an antiparallel b -strand 2form along b -hairpinloop. by helix 3. Helix 3, which lies near thebase of the sub- This loop and parallel b -strands 3and 4, which are unit and runs nearly parallel to theedge of the hexamer, separated by helix 1, form the first large b -sheet, which carries thesecond catalytic residue, Lys722.Strand 9 lies in aplane aligned with the sixfold axis. The distal returns along helix3,followed by ashort 3 10 helix 4, surface of this sheet forms the interface with the adjacent a -helix 5, andthen parallelstrand10. Strands6,9,and 10 subunits in the ring. Adisulfide bridge betweenCys617 and form athird small b -sheet, sandwiched by helix 3and Cys691 connects the end of helix 2tothe end of b strand 2, C-terminalhelix 6. The last nine residues adopt an stabilizing the subdomain. This unusualsurface-exposed extended conformation in only one molecule, while they disulfide bond is uniqueonly to Lon proteases from closely are disordered in theother ones. related enteric . At the base of the subdomain, Only afew deviations from the secondary structure of strand 5forms asecond, small b -sheet producing ashallow LonA have been reported forLonB. An isolated P-domain concavity toward the center of the ring. Strand 5is of AfLonB (residues 417–621, Fig. 3A) (Botos et al. connected to helix 2byaloop that containsthe catalytic 2005) contains an additional strand S0 on its Nterminus, Ser679. as well as along C-terminalhelix 7. An equivalent helix Following helix 2, arandom coil forms abridge to the is notpresent in the otherwise related MjLonB(residues second subdomain. Ashort b -strand 6leads into another 456–649, Fig.3A) (Imetal. 2004). In thelatter structure,

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helix 2, immediately adjacent to the part of the active site theD508A mutant does not change significantly the that contains the catalytic Ser550, is elongated (Fig. 3B), positions of either Lys552 (which is still wellordered leading to themodifications of the active sitethat will be and makes hydrogen bondstoThr534 and Gly547) o r further discussed below. Ser509, forwhich only asingleorientation,hydrogen bonded to awater molecule,isobserved. The active site of the P-domain Enzymatic mechanism at the proteolytic active site The firststructuredeterminedfor anyP-domain of Lon was of theinactive S679A mutantof EcLonA (Botos et al. Biochemical, mutational, and structural data have all 2004b); thus, the mutual disposition of theside chainsof establishedthatthe Ser-Lys dyad is responsible forthe thecatalyticdyadresiduesSer679 andLys722could only catalytic activity of proteases that belong to theLon be modeled.Inthe experimentally determinedstructure, family.However, in the absence of structuraldata de- Lys722 makes ahydrogen bondtothe carbonyloxygen scribing asubstrate bound in the active site of Lon, it is of Gly717 (Fig. 3B), whichisstrictly conserved in LonA necessarytoexamine the structures of other proteins that and LonB proteins, in addition to also interacting with usesuch adyad in order to model themechanism of either abound sulfate or theCterminus of another action of Lon. Relatedenzymes include E. coli type1 monomer. Theposition of thehydroxyl group of Ser679 signal peptidase(SPase) (MEROPS, clanSK; PDBcodes was modeled by makingminor adjustments to bringthe 1kn9, 1b12)(Paetzel et al.2002); theautoproteolytic N z atom of Lys722 to within ; 3A˚ of O g 1ofThr704 and enzymesLexA(1jhc, 1jhe) (Luoetal. 2001)and UmuD OofGly717. This model suggested an important role for (1umu) (Peat et al.1996); and l cI protein (1f39) (the Thr704,which belongs to astrictly conserved Tyr-Gly latter allbelonging to the MEROPS clan SF)(Bell et al. pairlocated 25 residues downstream of thecatalytic 2000). Acomparative analysisofconsensus sequences serine (Fig. 3B). Theresulting distance between Ser679 surrounding the catalytic residues of these peptidehydro- O g andLys722Nz was predicted to be ; 2.8 A ˚ . lases, as well as LonA and LonB proteases,revealed only Important questions about therelevant disposition of remote similarity betweenLons and LexA (Rotanova the residues in thecatalytic center of Lon proteases have 2002). At thesame time, structural comparisons reveal been raised on the basis of the arrangement of theactive theoverall similarity among thefolds of these proteins site of the isolated P-domain of MjLonB (Im et al. 2004). in the vicinity of their catalytic centers (Fig. 4). Also, in In this structure, the catalytic dyad formed by Ser550 those structures, the catalyticserine and lysine residues andLys593 (Fig.3B) was observed (distances of 3.0A˚ are thought to be in an ‘‘active’’ configuration, because between O g of the former andNz of the latter in thetwo they overlap very well with the catalytic residues of independent molecules in theasymmetric unit). In addi- TEM1 b -lactamase, arelated with aSer-Lys tion,athirdresidue, Asp547, was foundwithin hydrogen catalytic dyad. Although theP-domain of Lon has bonding distance of Lys593 (distances2.7 and3.3 A ˚ ). acompletely different overall fold, thestretch of residues Thedistances between Lys593and Thr575 were ; 3.4 A ˚ . that includes strand 5with thecatalytic Ser679 ( EcLon) These observations led to apostulate that thedetails of thecatalyticmechanism mightbedifferentbetween the two Lonsubfamilies (Im et al. 2004). Furtherdeparture from thepredicted arrangement of theactive site residues wasobserved in theatomic- resolution (1.2 A ˚ )structure of AfLonB(Botosetal. 2005), which differed from both EcLonA and MjLonB. Theside chain of Ser509 points outintothe solventand its O g group occupies twopositions, onewithin2.5 A ˚ of awater molecule, andanother 3.27 A ˚ away from O e 1of Glu472, aresidue that is strictly conserved amongall knownLonBproteases other than MjLon (Fig. 3B). AfLon Lys552 is very well ordered andits N z groupmakes three hydrogen bonds, being2.77A˚ away fromthe carbonyl oxygen of Gly547, 2.88A˚ away from O g 1ofThr534, and 2.69 A ˚ from O d 1ofAsp508. The O d 2group of Asp508 ˚ Figure 4. Active site superposition of Lon with other serine proteases: makesanother hydrogen bondtoOg 1ofThr534 (2.64 A ) E. coli Lon protease (green; PDB code 1rr9); subtilisin complexed with and accepts abondfrom themain chain amide nitrogenof eglin (cyan; PDB code 1cse); l cI (magenta; PDB code 1f39); umuD Gly535 (2.99A˚ ). Removalofthe sidechain of Asp508 in (yellow; PDB code 1umu); LexA (gray; PDB code 1jhe).

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andthe beginning of helix 2can be superimposed quite foundinthe structures of the EcLonAP-domain, the l cI well onto thecorresponding segments in the four struc- protein, and UmuD 9 ;wenote that thelatter structure is tures listed above.This alignmentalsobrings thecatalytic lacking thepolypeptide segment with the scissile bond. lysines into excellent superposition. Thewater makes an additionalhydrogen bondtothe Comparative analysisofthe active sites of these carbonyl oxygen of theresidueatthe third position enzymes identifies athird residue that mightassist the N-terminal to the catalytic serine (Asp676 in the case of Ser-Lys dyad during catalysis. This residue is either EcLonA). Considering the similarity of the active site aserine or athreonine, with the side chain O g located configurationsofthe P-domain andthe other enzymes, it within hydrogen-bondingdistance of the catalyticlysine. was possible that theamide nitrogens of Ser679 andthe Therole of such aresidu e(Ser278 in SPase,Thr154in preceding residue, Pro678, together formed the oxyanion LexA,Thr704in EcLonA,Thr534 in AfLonB, and hole in Lon. However, as proline hasnot been shownto Thr575in MjLonB) might be similar to that of the participate in the formation of oxyanion holes in other aspartate present in the classic of serine enzymes, itsability to servethis function in Lonisrather proteases (Dodson andWlodawer1998). However, it questionable.Asecond candidatewould be the amide is noteworthy that mutant forms EcLonT704A and nitrogen of thenearby Trp603,which contributes to a AfLonT534A retain significant proteolytic activity.Thus, highly conserved motif, GLAW/Y in LonAs or GLAV/I afullrole of these threonine residues forthe function of in LonBs, for which no functionhas yet been identified. Lon proteases needs to be clarifiedinfurther studies. Do the LonBsubfamily enzymes have adifferent Oligomeric structure of Lon proteins mechanismofaction as postulatedbased on the structural studies on MjLonB (Im et al.2004)? In that structure, Isolatedforms of both Aand Btype Lon proteasesare Asp547 formed asalt bridge with Lys593 (Fig. 3B), oligomeric, butthe stoichiometry of the subunits, and suggesting that it directly impinges on acatalytic residue whetherthe stoichiometry is thesame in all Lonspecies, andcould be required foractivity.However, Asp547 is have notbeen established.The stoichiometry was notuniversally conserved in allB-type Lons; forexam- reported to be sevensubunits in yeast mitochondrial ple, Glu506 is presentatthe equivalentposition in Lon basedonanumber of independent criteria, including AfLonB, andmutation of thelatter residue to alanine electron microscopic imageanalysis (Stahlbergetal. did not significantlyinfluence theenzymatic activity 1999). For EcLon, sedimentation data pointed to two (Botos et al. 2005).Itispossible that thestructure states putatively composed of fourand eight subunits observed for MjLonBrepresents an alternative state of (Goldbergetal. 1994), whereas for M. smegmatis Lon, LonBthatcan undergo achange to theactive configura- an equilibrium between hexamer/tetramer/dimer or tion, butfurther data are needed to address the question of hexamer/trimer was reported (Rudyak et al.2001). STEM the role of this aspartate residue. Giventhe significant analysis of intact EcLon andanN-domain-deletedform degree to whichthe details of theactive site differ of Lon (residues 309–784)showed predominantlyhex- substantially in the structures of theP-domains solved americ assemblies or,for intact Lon only,complexes to date, it maybethat none fully representsthe active of two associated hexamers (FSR, MRM; M. Kessel, enzyme. In addition, theactive site of the viral protein R. Leapman,unpubl.). Very recently,negative stain VP4 in which theactive site serine was present(Feldman electron microscopy of EcLongaveparticles that pro- et al.2006) is in excellent agreement with the structure duced averaged images with sixfold symmetry,also of EcLonA. suggestingthat EcLon forms ringswith sixsubunits(Park An additional structural feature, the‘‘,’’ et al. 2006). Bacillus thermoruber Lon wasreportedtobe is critical for enzymatic activity in serine proteases, ahexamer basedonacombination of gel filtration and because it helps to stabilize the formation of atetrahedral sedimentation velocity experiments andoncross-linking intermediate during catalysis (Kraut 1977). In the LexA of intact and truncated species (Lee et al.2004). Finally, mutant1jhe, theoxyanion hole was identifiedonthe basis it wassuggested that the oligomeric state of EcLoncould of thehydrogen-bonded interactions of the carbonyl be modulated by reaction conditions in vitro (Vineyard oxygen of Ala84, which formsthe scissile bond targeted et al.2005),aswellasbyvarious cellular events in vivo during autoproteolysis(Luoetal. 2001). Twomain chain (Nishii et al.2005). amide nitrogens, onefrom the catalytic serine and the The crystalstructures of the EcLonA and AfLonB other from thepreceding residue, form theoxyanion hole. P-domainshaveprovided data favoringhexameric ring In threeother structures of LexA in which the loop structuresfor these enzymes. In the EcLonA P-domain containingthe Ala84-Gly85 bond is not in aconformation crystals (Botosetal. 2004b), as well as oneofthe crystal allowing self-cleavage,awater molecule occupies the forms of AfLonB P-domain (Botos et al. 2005), the position of theAla84 carbonyl. An equivalentwater is asymmetricunit contained sixmolecules related by an

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approximatesixfold non-crystallographic symmetry P-domain (Botos et al. 2005), which is present in solution (NCS)axis, revealing ahexamer in which individual as amonomer,but its oligomeric state in crystals differs monomersformaring (Fig.5). Viewed from theside, the among the four reported forms. In two of them, orthorhom- ring is dome-shaped,with adiameter of ; 100A˚ at the bic and monoclinic, the asymmetric units contain hexamers base and ; 50 A ˚ at the top. Asolvent-accessible central very similar to the one described abovefor EcLonA. pore ; 32 A ˚ long runsthrough thehexamer.Ithas Charge distribution on the surface of the hexamer of adiameter of ; 18 A ˚ at theentrance from theproximal AfLonB P-domainhas very similar characteristics to the side and widens slightlytoward the distal end. The pore distribution described for EcLonA. However,two related entrancefromthe proximal surface(16–18 A ˚ )issignifi- hexagonal crystal forms found for the wild-type enzyme cantlylargerinsize than the entrychannel in E. coli and for the D508Amutant haveonly asingle molecule in ClpP ( ; 10 A ˚ )(Wang et al. 1997) and could be expected the asymmetric unit, with the molecules following ahelical to accommodate twofolded a -helices or b -strandsor packing pattern along the 6 5 axis. Translation of the unstructured loops with two polypeptide chains. Anum- molecules in steps of ; 6.5 A ˚ alongthe hexagonal axis berofnegatively chargedand polar residues arelocated yields ahexameric ring that superimposesperfectly on the towardthe distal endofthe pore, making the distal end hexameric rings present in the orthorhombic and mono- rather negatively charged, while theproximal half of the cliniccrystal forms. These observations suggest that the pore has several positively charged residues, which might hexameric ringisthe favored configuration at high protein serveagating function forsubstrate entry. concentrations,with the caveat that the contactsfavored The presence of hexamericsymmetryinseveralcrystal under crystallization conditions may differ slightly from forms of EcLonA P-domainssuggests that thehexameric thoseinthe intact oligomer under more physiological ring assembly is not an artifact of crystallization but conditions. Since, in most AAA+ proteins, the major ratherabiologically significant unit. Interactions between contacts stabilizing the oligomeric states are madethrough isolated P-domainsare weak; EcLonA P-domainsare mono- the AAA+ modules themselves, it is likely that P-domain meric in solution at concentrations as high as 100 m M interactions are strongestwhenthey are broughtinto close subunit equivalents. No higher order oligomers were proximity by assembly of the AAA+ domains.Quite reported in the structure of MjLonB P-domain (Im et al. possibly,contacts betweenP-domains may be somewhat 2004), in which the two molecules in the asymmetric unit variable and responsive to the conformational state of the formed alooseisologously bonded dimer.Inaddition, AAA + domain. amore complicated picturewas observed for the AfLonB P-domain interactions may also influence the confor- mation andactivity of the A-domain, which could explain theproperties of some previously isolated mutants. In earlierattempts to identify catalyticLon residues, the very highly conserved His665 andHis667 were mutated, and both mutantenzymes lack protein-degrading activity (Starkovaetal. 1998). These mutants also caused a90% reduction in ATPase activity,suggesting that they per- turbed communication betweenthe P- andA-domainsor indirectly altered thestructureofthe A-domain. In the P-domain crystal, His665 andHis667 both lieatthe olig- omeric interface. Their mutation should alterthe subunit interactions, which in turn couldperturbthe interactions betweenthe A-domains or couldalter the timing of con- formationalchanges in theA-domain. Oneofthe earliest described mutants of E. coli Lon, called CapR9 because it caused achangeinproductionofthe colanic acid capsular polysaccharide (Hua andMarkovitz 1972), was shown to have asingle point mutation in Glu614 (Oh et al.1998). Lon-E614Kisadominant-negative mutant, because it can form mixedoligomers with wild-type Lon andinterfere with its activity.Inthe P-domain crystal, theside chain of Glu614 liesatthe interfacebetween subunits and makes Figure5. Oligomeric structure of the P-domain of LonA. Top(A )and asalt bridge with Arg710from theadjacent subunit. side ( B )views of the EcLonA hexamers as seen in the crystals are colored Glu614 is present in virtually allbacterialLon proteases, accordingtocharge distribution: (blue) positive areas, (red) negativeareas. andArg710 is highly conservedorisreplacedbyother

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positively chargedorhydrophilic residues.Inthe crystal, theinteractions seen in the two-component HslUV. As in Glu614also makes ahydrogen bond with His667ofthe HslUV,the interaction betweenthe AAA+ module and the samesubunit andthusaffects subunit bonding by inter- protease in Lon is symmetrical andismediated through acting with this residue as well.The region around alinear polypeptidelinkage, although thecovalent Glu614, His667, andHis665containsfivestructural linkagebetween thedomains in Lon perhaps allows more water molecules,indicating that perturbationofany of directmechanical transductionofconformational changes these residues could perturb asignificant part of the in the twodomains in thesubunit. Moreover, intersub- subunitinterface. Thecorrelationbetween the mutational unit domain–domain interactionsbetween ATPase and data and themodel placing these residues at thesubunit proteolytic sites were revealed in EcLonbycomplemen- interfacesuggests that themodel of theP-domain i nthe tation of twomutant forms, LonK362Q and LonS679A crystal is quite likely to be biologically significant. (Melnikovetal. 2001; Tsirulnikovetal. 2003). When the a -domain structure of Lonismodeled onto thehexamer of HslU, theC-terminal endofthe AAA+ module is Functional insights from models of full-lengthLon predicted to lie on the ring surface proximal to the The recently acquired structuraldatacan nowbecom- P-domain. The Cterminusofthe a -domainislinkedto bined with the structures of otherATP-dependent pro- the P-domain by ashort length of polypeptide, which teases to assemble aworking model of howLon might defines theorientationofthe protease domain with look andwork. Most AAA+ proteins form hexameric respecttothe AAA + module.Inthe proposed model rings in which the small a -domain interacts with the of the combined Lon A- andP-domains (Lon-AP), we a / b -domain of theadjacent subunit.These assemblies assume that the P-domain structure is correct with respect are stabilized by ATPbinding, and in many cases ATP to theposition of its N-terminal peptide. This assumption hydrolysis requires the assembled hexamer state. The is justified because that portionofthe structure is well nucleotidebinds in acrevice formed by the a -and a / b - determined in all themodels constructed so farand domains from onesubunit, butatleast one residue, because thepeptide is stabilized by numerousinteractions usuallyanarginine (referred to as an ‘‘Argfinger’’), from with thefolded domain. These restraints place the the adjacent subunit protrudesintothe nucleotidebinding P-domainhexamer with the domed surface abutting the site and canactivatenucleotide hydrolysis.Inthe case AAA+ module andthe concavesurface of the P-domain of assembliesinwhich the functional partner is expressed distal to the AAA+ module. Assuming Lon domains are independently, interaction between the AAA+ hexamer disposed as in other AAA+ proteins, theN-domain of Lon and thefunctional partner is mediatedbyanextension would be positioned on or near the opposite face of the from the AAA + domain that fits into aspecific docking AAA+ module,where they wouldassistinthe recruitment groove on thefunctional component. In theHslUV of substrates to binding sites on theproximalringsurface complex, theC-terminal heptapeptide of HslU penetrates or within the axial channel. Bound substrates would then adeep depressioninthe surface of HslV,anchoring the be translocated through theAAA+ module to theprotease. two hexamers together (Seong et al. 2002). In isolated Themodel hasafew expected, as well as some unex- HslU, theC-terminal peptide is folded back anddocks pected, features. Theaxial pore described abovewould into ahydrophobic groove on HslU itself (Bochtler et al. alignwith the exit pore from the AAA + hexamer, 2000), suggesting that anucleotide-regulated conforma- allowinganunfoldedpolypeptide or unstructured loop tional change is required to flip out the docking peptide to pass directly from the AAA + modulethroughthe pore, to allowittointeract with HslV.InClp proteases, aloop whichwould then bring it into contact with the pro- withahighlyconserved motif at its apexprotrudes from teolytic active sites. One surprise is that the active sites theAAA+ moduleand docks into agroove on thesurface are not sequestered in this model, butlie in asolvent- of ClpP.Despiteasymmetry mismatch between hexame- exposed shallowcrevice on the distal surface of the pro- ricClpA or ClpX andheptameric ClpP,which would teasering. In ordertoassure processive protein degrada- prevent all the‘‘ClpPloops’’from being utilized simul- tion with this arrangement, movement of the substrate taneously,the nucleotide-promoted interaction between protein from theAAA+ module through the protease the functional partners is quitetight(Kd <10nM). One wouldneed to be rate-limiting andtimed to allowasuf- proposal regarding themismatch between thecomponents ficient number of cleavage events to generate the small in Clp proteases is that it allows flexibility and relative peptides that are theusual productofdegradationbyLon. movement between thecomponents during thecatalytic This configuration also raises apuzzling question cycles (Beuron et al.1998). regardinghow proteins in the surrounding milieu avoid Lon, in whichthe protease domain is apolypeptide being degraded, since theLon active sites aredirectly extension joined to theAAA+ module, presents yet accessible from thedistal surface. One possibility is that, another arrangement, butitmightresemble more closely although the active sites areaccessible, the catalytic

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residues are notconfigured properly forproteolysis betweenunliganded Lon and Lon withATP bound, which withoutsomesignalfromth eAAA+ module or from could reflect aloosening or openingofcontacts between aprotein within the axial channel.Such acondition exists subunits in aring (Stahlbergetal. 1999). Nicking of the in theHslUVsystem, in whichitwas shownthat the protein would destabilize itstertiary structure, allowing isolated C-terminal decapeptide of HslU canallosteri- thesubstrate to be encapsulatedwhen the rings come callyactivatepeptide cleavagebyHslV(Seongetal. back together,leading to processive unfoldingand trans- 2002), implying thatthe proteolyticactivity of HslV is location intothe proteolytic sites. An alternative possi- dependentonHslUnot only fordelivery of substrates but bility is that Lon canunfold aproteinfromthe middleand also for regulating that catalytic activity of the active site. translocateapolypeptideloop or multiple polypeptide Making thefunctionality of the active sites dependent segments. Thesize of the axialchan nel is notnecessarily on allosteric effects produced by substrates bound on the fixed. Gating of theaxial channel hasbeen confirmed for AAA+ module or within thechannel would provide theproteasome, in whichthe channelscan undergo protectionfromunwanted cleavage of non-substrate amajor rearrangementinresponse to binding of an proteins. Thepropertiesofthe mutant, Lon-D676N, can activator protein(Groll et al. 2000; Whitby et al.2000), be interpreted in terms of allosteric communication and recent data on ClpP indicatethat theN-terminal between theaxial channel of the P-domain andthe peptide canadoptdifferent configurations that alter the ATPase active sites of Lon. Lon-D676N is completely size and characterofthe axialchannel (Kangetal. 2004; inactive forprotein degradation; it retains some basal Bewleyetal. 2006). Aquestion raised by theopenness of ATPase activity,but no activationofATPaseactivity theproteolytic sites in theP-domaincrystals is whether occurs upon bindingofprotein substrates. Asp676 lies at folded substratescan directly access the proteolytic sites. the distal endofthe axial channel andstabilizes an a / b - Theinitial cleavage couldresultinstructural destabiliza- turnmadebyhelix 1and strand 3, which together make tion,allowingthe A-domain to engage theprotein, further up the walls of the axial channel. We propose that, in unfold it, and translocate it to the proteolytic domain to Lon-D676N, destabilization of thechannel blocks sub- complete thedegradation.Itisnot clear in this mecha- strate passage, which in turn preventsac tivationofthe nismhow release of the nicked protein would be avoided ATPase activity,which is needed to couple movement of to ensure processive degradation, butthe proximity to the thesubstrate through the channel with additional rounds A-domain or N-domains mightbesufficient to permit of ATPhydrolysis. efficient retention of the nicked protein. Furtherinsight Recent data obtained with mitochondrial Lon suggest into Lon’smechanism of action must awaitadditional that some substratesbindLon andcan be actedonbyLon structural and functional studies,but it is likely that the while they are stillfolded (Ondrovicova et al. 2005). The diversity of ATP-dependent proteases reflects thewide proteins, the a -subunit of the mitochondrial processing variation in substrates targeted fordegradation andthat proteaseand steroidogenic acute regulatory protein, are themechanisms by which theyoperate areoptimized for degraded by Loninvitro under conditionsinwhich they thetypesofsubstratestheyselectand thecellular are resistant to trypsin digestion andfoldedinfunction- conditions under which theymust function. The ability ally competent states.The initial peptide bonds cleaved of Lon active sites to switch betweenlatent and active are at sites well away from the ends of the polypeptide, configurations might evenprovide ameans by which and arepresent in the three-dimensional structure in metabolic effectors could activate more promiscuous regions exposed on the surface in the folded proteins. proteolyticactivity under exigent circumstances. Re- Subsequentdegradative steps occurprocessively from cently,ligand-induced stimulation of indiscriminate pro- those sites. These data clearly indicate that polypeptide teolytic activity was proposedfor ClpP (Brotz-Oesterhelt loops can gain access to theproteolytic sites of Lonand et al. 2005), anditispossible that such activityis thatLon does notneedtounravel aproteinfromthe Nor expressedbyLon and otherproteolytic componentsof Ctermini. In fact,studies with fusion protein substrates ATP-dependent proteases under stress conditions or when suggestthat yeast Lon hasarelatively poor ability to damagedorunfolded proteinsaccumulate. unravelproteins andisonly able to degradeproteins that have unstable tertiary structure (von Janowsky et al. Conclusions 2005). Howdothese data affect models of ATP-dependent Although Lonswere the first ATP-dependent proteases to degradation by Lon? It was suggested by Ondrovicova be studied, our knowledgeoftheir structuraland bio- et al. (2005)thatLon rings might open to allowthe folded chemical properties haslaggedbehind those of other protein into theinterior of theAAA+ module and into similarly regulated proteolytic enzymes. In the absenceof the proteolytic sites. Studies of yeast Lon by electron acrystal structure of afull-length enzyme, some impor- microscopy indicate large conformational differences tant questions must remain unanswered. Nevertheless, the

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recently solved structures of individual Londomains, Atomic-resolution crystal structure of the proteolytic domain of Archae- oglobus fulgidus Lonreveals the conformational variability in the active coupled withbiochemical, biophysical, andelectron sites of Lon proteases. J. Mol. Biol. 351: 144–157. microscopy data, areproviding ourfirst insights into Brotz-Oesterhelt, H., Beyer,D., Kroll, H.P., Endermann, R., Ladel, C., howthe structural features of Lon relate to its functions Schroeder,W., Hinzen, B., Raddatz, S., Paulsen, H., Henninger,K., et al. 2005. Dysregulation of bacterial proteolytic machinery by anew class of and activities. Already there are intriguing hints of antibiotics. Nat. Med. 11: 1082–1087. differences in mechanisms of action andmodes of Burton, R.E., Siddiqui, S.M., Kim, Y.I., Baker,T.A., and Sauer,R.T.2001. Effects of protein stability and structure on substrate processing by the regulation between Lonand other ATP-dependentpro- ClpXP unfolding and degradation machine. EMBO J. 20: 3092–3100. teases.Giventhe important biological rolesplayedby Choi, K.H. and Licht, S. 2005. Control of peptide product sizes by the energy- Lon proteases, it is ourhope that the still fragmentary dependent protease ClpAP. Biochemistry 44: 13921–13931. Chung, C.H. andGoldberg, A.L. 1981. The product of the lon (capR) gene in informationpresented in this review will provide abasis Escherichia coli is the ATP-dependent protease, protease La. Proc. Natl. for more detailed studies on the structure and function Acad. Sci. 78: 4931–4935. DeLaBarre, B. and Brunger,A.T.2005. Nucleotide dependent motion and of thisubiquitous familyofenzymes. mechanism of action of p97/VCP. J. Mol. Biol. 347: 437–452. Dodson, G. and Wlodawer,A.1998. Catalytic triadsand their relatives. Trends Biochem. Sci. 23: 347–352. Ebel, W.,Skinner,M.M., Dierksen, K.P., Scott, J.M., and Trempy,J.E. 1999. A Acknowledgments conserved domain in Escherichiacoli Lon protease is involved in substrate discriminator activity. J. Bacteriol. 181: 2236–2243. This workwas supported in part by agrant from theRussian Feldman, A.R., Lee, J., Delmas, B., and Paetzel, M. 2006. Crystal structure of Foundation for Basic Research (project no.05-04-48383) to anovel viral protease with aserine/lysine catalytic dyad mechanism. T.V.R, by the U.S. Civilian Research andDevelopment Foun- J. Mol. Biol. 358: 1378–1389. dation granttoT.V.R.and A.W.(project no.RB1-2505-MO-03), Goldberg, A.L. 1992. The mechanism and functions of ATP-dependent proteases in bacterial and animalcells. Eur.J.Biochem. 203: 9–23. and by theIntramural Research Program of theNIH, National Goldberg, A.L., Moerschell, R.P., Chung, C.H., and Maurizi, M.R. 1994. ATP- Cancer Institute, Center for Cancer Research. dependent protease La (lon) from Escherichia coli. Methods Enzymol. 244: 350–375. Gottesman, S. 1996. Proteases and their targets in Escherichia coli. Annu. Rev. References Genet. 30: 465–506. ———. 2003. Proteolysis in bacterial regulatory circuits. Annu. Rev.Cell Dev. Amerik, A.Y., Chistyakova, L.G., Ostroumova, N.I., Gurevich, A.I., and Biol. 19: 565–587. Antonov,V.K. 1988. Cloning, expression and structure of the functionally Gottesman, S. and Maurizi, M.R. 1992. Regulation by proteolysis: Energy- active shortened lon gene in Escherichia coli. Bioorg. Khim. 14: 408–411. dependent proteases and their targets. Microbiol. Rev. 56: 592–621. Amerik, A.Y., Antonov,V.K., Ostroumova, N.I., Rotanova, T.V.,and Groll, M., Ditzel, L., Lowe, J., Stock, D., Bochtler,M., Bartunik, H.D., and Chistyakova, L.G. 1990. Cloning, structure and expression of the full lon Huber,R.1997. Structure of 20S proteasome from yeast at 2.4 A ˚ resolution. gene of Escherichia coli coding for ATP-dependent protease La. Bioorg. Nature 386: 463–471. Khim. 16: 869–880. Groll, M., Bajorek, M., Kohler,A., Moroder,L., Rubin, D.M., Huber,R., Amerik, A.Y., Antonov,V.K., Gorbalenya, A.E., Kotova, S.A., Rotanova, T.V., Glickman, M.H., and Finley,D.2000. Agated channel into the proteasome and Shimbarevich, E.V. 1991. Site-directed mutagenesis of La protease. core particle. Nat. Struct. Biol. 7: 1062–1067. Acatalytically active serine residue. FEBS Lett. 287: 211–214. Groll, M., Bochtler,M., Brandstetter,H., Clausen, T.,and Huber,R.2005. Baumeister,W., Walz, J., Zuhl, F.,and Seemuller,E.1998. The proteasome: Molecular machines for protein degradation. ChemBioChem 6: 222–256. Paradigm of aself-compartmentalizing protease. Cell 92: 367–380. Guo, F.,Maurizi, M.R., Esser,L., and Xia, D. 2002. Crystal structure of ClpA, Bell, C.E., Frescura, P.,Hochschild, A., and Lewis, M. 2000. Crystal structure an Hsp100 chaperone and regulator of ClpAP protease. J. Biol. Chem. 277: of the l repressor C-terminal domain provides amodel for cooperative 46743–46752. operator binding. Cell 101: 801–811. Hegerl, R., Pfeifer,G., Puhler,G., Dahlmann, B., and Baumeister,W.1991. The Beuron, F.,Maurizi, M.R., Belnap, D.M., Kocsis, E., Booy,F.P., Kessel, M., and three-dimensional structure of proteasomes from Thermoplasma acidophi- Steven, A.C. 1998. At sixes and sevens: Characterization of the symmetry lum as determined by electron microscopy using random conical tilting. mismatch of the ClpAP chaperone-assisted protease. J. Struct. Biol. 123: FEBS Lett. 283: 117–121. 248–259. Hoskins, J.R., Pak, M., Maurizi,M.R., and Wickner,S.1998. The role of the Bewley,M.C., Graziano, V.,Griffin, K., and Flanagan, J.M. 2006. The ClpA chaperone in proteolysis by ClpAP. Proc. Natl. Acad. Sci. 95: 12135– asymmetry in the mature amino-terminus of ClpP facilitates alocal 12140. symmetry match in ClpAP and ClpXP complexes. J. Struct. Biol. 153: Hua, S.S. and Markovitz, A. 1972. Multiple regulator gene control of the 113–128. galactose operon in Escherichia coli K-12. J. Bacteriol. 110: 1089–1099. Bochtler,M., Ditzel, L., Groll, M., and Huber,R.1997. Crystal structure of Im, Y.J., Na, Y.,Kang, G.B., Rho, S.H., Kim, M.K., Lee, J.H., Chung, C.H., and heat shock locus V(HslV) from Escherichia coli. Proc. Natl. Acad. Sci. Eom, S.H. 2004. The active site of aLon protease from Methanococcus 94: 6070–6074. jannaschii distinctlydiffers from the canonical catalytic dyad of Lon Bochtler,M., Hartmann, C., Song, H.K., Bourenkov,G.P., Bartunik, H.D., and proteases. J. Biol. Chem. 279: 53451–53457. Huber,R.2000. The structures of HslU and the ATP-dependent protease Iyer,L.M., Leipe, D.D., Koonin, E.V., and Aravind, L. 2004. Evolutionary HslU-HslV. Nature 403: 800–805. history and higher order classification of AAA+ ATPases. J. Struct. Biol. Bota, D.A. and Davies, K.J. 2002. Lon protease preferentially degrades 146: 11–31. oxidized mitochondrial aconitase by an ATP-stimulated mechanism. Nat. Kang, S.G., Maurizi, M.R., Thompson, M., Mueser,T., and Ahvazi, B. 2004. Cell Biol. 4: 674–680. Crystallography and mutagenesis point to an essential role for the Botos, I., Melnikov,E.E., Cherry,S., Khalatova, A.G., Rasulova, F.S., N-terminus of humanmitochondrial ClpP. J. Struct. Biol. 148: 338–352. Tropea, J.E., Maurizi,M.R., Rotanova, T.V.,Gustchina, A., and Kenniston, J.A., Baker,T.A., Fernandez, J.M., and Sauer,R.T.2003. Linkage Wlodawer,A.2004a. Crystal structure of the AAA+ a domainof E. coli between ATPconsumption and mechanical unfolding during the protein Lon protease at 1.9 A ˚ resolution. J. Struct. Biol. 146: 113–122. processing reactions of an AAA+ degradation machine. Cell 114: 511–520. Botos, I., Melnikov,E.E., Cherry,S., Tropea, J.E., Khalatova, A.G., Kim, D.Y.and Kim, K.K. 2003. Crystal structure of ClpX molecular chaperone Rasulova, F.,Dauter,Z., Maurizi, M.R., Rotanova, T.V.,Wlodawer,A., from Helicobacter pylori. J. Biol. Chem. 278: 50664–50670. et al. 2004b.The catalytic domain of Escherichia coli Lonprotease has Kim, Y.I., Burton, R.E., Burton, B.M., Sauer,R.T., andBaker,T.A. 2000. aunique fold and aSer-Lys dyad in the active site. J. Biol. Chem. 279: Dynamics of substrate denaturation and translocation by the ClpXP 8140–8148. degradation machine. Mol. Cell 5: 639–648. Botos, I., Melnikov,E.E., Cherry,S., Kozlov,S., Makhovskaya, O.V., Kisselev, A.F., Akopian, T.N., Woo, K.M., and Goldberg, A.L. 1999. The Tropea, J.E., Gustchina, A., Rotanova, T.V.,and Wlodawer,A.2005. sizes of peptides generated from protein by mammalian 26 and 20 S

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Structural featuresofLon proteases

proteasomes. Implicationsfor understanding the degradative mechanism Rasulova, F.S., Dergousova, N.I., Melnikov,E.E., Ginodman, L.M., and and antigen presentation. J. Biol. Chem. 274: 3363–3371. Rotanova, T.V. 1998a. Synthesis and characterisation of ATP-dependent Kraut, J. 1977. Serine proteases: Structure and mechanism of catalysis. Annu. forms of Lon-proteinase with modified N-terminal domain from Escher- Rev. Biochem. 46: 331–358. ichia coli. Bioorg. Khim. 24: 370–375. Krzywda, S., Brzozowski, A.M., Verma, C., Karata,K., Ogura, T.,and Rasulova, F.S., Dergousova,N.I., Starkova, N.N., Melnikov,E.E., Rumsh, L.D., Wilkinson, A.J. 2002. The crystal structure of the AAA domain of the Ginodman, L.M., and Rotanova, T.V. 1998b.The isolated proteolytic ATP-dependent protease FtsH of Escherichia coli at 1.5 A ˚ resolution. domainof Escherichia coli ATP-dependent protease Lon exhibits the Structure 10: 1073–1083. peptidase activity. FEBS Lett. 432: 179–181. Langer,T.2000. AAA proteases: Cellular machines for degrading membrane Rawlings, N.D., Tolle, D.P., and Barrett, A.J. 2004. MEROPS: The peptidase proteins. Trends Biochem. Sci. 25: 247–251. database. Nucleic Acids Res. 32: D160–D164. Lee, A.Y., Hsu, C.H., and Wu,S.H. 2004. Functional domains of Brevibacillus Reid, B.G., Fenton, W.A., Horwich, A.L., and Weber-Ban, E.U. 2001. ClpA thermoruber lon protease for oligomerization and DNAbinding: Role of mediates directional translocation of substrate proteins into the ClpP N-terminal and sensor and substrate discrimination domains. J. Biol. protease. Proc. Natl. Acad. Sci. 98: 3768–3772. Chem. 279: 34903–34912. Rotanova, T.V. 2002. Peptide with catalytic dyad Ser-Lys. Similarity Lenzen, C.U., Steinmann, D., Whiteheart, S.W., and Weis, W.I. 1998. Crystal and distinctions of the active centers of ATP-dependent Lon proteases, structure of the hexamerization domain of N-ethylmaleimide-sensitive LexA repressors, signal peptidases and C-terminal processing proteases. fusion protein. Cell 94: 525–536. Vopr.Med. Khim. 48: 541–552. Li, M., Rasulova, F.,Melnikov,E.E., Rotanova, T.V.,Gustchina, A., Rotanova, T.V.,Melnikov,E.E., and Tsirulnikov,K.B. 2003. Catalytic dyad Maurizi, M.R., and Wlodawer,A.2005. Crystal structure of the N-terminal Ser-Lys at the active site of Escherichia coli ATP-dependent Lon-pro- domain of E. coli Lon protease. Protein Sci. 14: 2895–2900. teinase. Bioorg. Khim. 29: 97–99. Lowe, J., Stock, D., Jap, B., Zwickl, P.,Baumeister,W., and Huber, R. 1995. Rotanova, T.V.,Melnikov,E.E., Khalatova, A.G., Makhovskaya, O.V., Botos, I., Crystal structure of the 20S proteasome from the archaeon T. acidophilum Wlodawer,A., and Gustchina, A. 2004. Classification of ATP-dependent at 3.4 A ˚ resolution. Science 268: 533–539. proteases Lon and comparison of the active sites of their proteolytic Luo, Y.,Pfuetzner,R.A., Mosimann, S., Paetzel, M., Frey,E.A., Cherney,M., domains. Eur.J.Biochem. 271: 4865–4871. Kim, B., Little, J.W., and Strynadka, N.C. 2001. Crystal structure of LexA: Roudiak, S.G. and Shrader,T.E. 1998. Functional role of the N-terminal region Aconformational switch for regulation of self-cleavage. Cell 106: 585– of the Lon protease from Mycobacterium smegmatis. Biochemistry 37: 594. 11255–11263. Lupas, A.N. andMartin, J. 2002. AAA proteins. Curr.Opin. Struct. Biol. 12: Roudiak, S.G., Seth, A., Knipfer,N., andShrader,T.E. 1998. The lon protease 746–753. from Mycobacterium smegmatis:Molecular cloning, sequence analysis, Martin, A., Baker,T.A., and Sauer,R.T.2005. Rebuilt AAA+ motors reveal functional expression, and enzymatic characterization. Biochemistry 37: operating principles for ATP-fuelled machines. Nature 437: 1115–1120. 377–386. Maurizi, M.R. andLi, C.C. 2001. AAA proteins: In search of acommon Rouiller,I., DeLaBarre, B., May,A.P., Weis, W.I., Brunger, A.T., molecular basis. International Meeting on Cellular Functions of AAA Milligan, R.A., and Wilson-Kubalek, E.M. 2002. Conformational changes Proteins. EMBO Rep. 2: 980–985. of the multifunction p97 AAA ATPase during its ATPase cycle. Nat. Struct. Melnikov,E.E., Tsirulnikov,K.B., andRotanova, T.V. 2001. Coupling of Biol. 9: 950–957. proteolysis to ATPhydrolysis upon Escherichia coli Lon protease func- Rudyak, S.G., Brenowitz, M., and Shrader,T.E. 2001. Mg2+-linked oligomer- tioning II. Hydrolysis of ATPand activity of the enzyme peptide hydrolase ization modulates the catalytic activity of the Lon(La) protease from sites. Bioorg. Khim. 27: 120–129. Mycobacterium smegmatis. Biochemistry 40: 9317–9323. Neuwald, A.F., Aravind, L., Spouge, J.L., andKoonin, E.V.1999. AAA+ :A Sauer,R.T., Bolon, D.N., Burton, B.M., Burton, R.E., Flynn, J.M., Grant, R.A., class of chaperone-like ATPases associated with the assembly,operation, Hersch, G.L., Joshi, S.A., Kenniston, J.A., Levchenko, I., et al. 2004. and disassembly of protein complexes. Res. 9: 27–43. Sculpting theproteome with AAA+ proteases and disassembly machines. Nishii, W.,Suzuki, T.,Nakada, M., Kim, Y.T.,Muramatsu, T.,and Cell 119: 9–18. Takahashi, K. 2005. Cleavage mechanism of ATP-dependent Lon protease Seong, I.S., Kang, M.S., Choi, M.K., Lee, J.W., Koh, O.J., Wang, J., Eom, S.H., toward ribosomal S2 protein. FEBS Lett. 579: 6846–6850. and Chung, C.H. 2002. The C-terminal tails of HslU ATPase act as Niwa, H., Tsuchiya, D., Makyio, H., Yoshida, M., and Morikawa, K. 2002. amolecular switch for activation of HslV peptidase. J. Biol. Chem. 277: Hexameric ring structure of the ATPase domain of the membrane-in- 25976–25982. tegratedmetalloprotease FtsH from Thermus thermophilus HB8. Structure Singh, S.K., Grimaud, R., Hoskins, J.R., Wickner, S., and Maurizi, M.R. 2000. 10: 1415–1423. Unfolding and internalization of proteins by theATP-dependent proteases Ogura, T. and Wilkinson, A.J. 2001. AAA+ superfamily ATPases: Common ClpXP and ClpAP. Proc. Natl. Acad. Sci. 97: 8898–8903. structure–diverse function. Genes Cells 6: 575–597. Sousa, M.C. and McKay,D.B. 2001. Structure of Haemophilus influenzae HslV Oh, J.Y., Eun, Y.M., Yoo, S.J., Seol, J.H., Seong, I.S., Lee, C.S., and proteinat1.9 A ˚ resolution, revealing acation- near the catalytic Chung, C.H. 1998. LonR9 carrying asingle Glu614 to Lysmutation site. Acta Crystallogr. D57: 1950–1954. inhibits the ATP- dependent protease La (Lon) by forming mixed Sousa, M.C., Trame, C.B., Tsuruta, H., Wilbanks, S.M., Reddy,V.S., and oligomeric complexes. Biochem. Biophys. Res. Commun. 250: 32–35. McKay,D.B. 2000. Crystal and solution structures of an HslUV protease– Ondrovicova,G., Liu, T.,Singh, K., Tian, B., Li, H., Gakh, O., Perecko, D., chaperone complex. Cell 103: 633–643. Janata, J., Granot, Z., Orly,J., et al. 2005. Cleavage site selection within Stahlberg, H., Kutejova,E., Suda, K., Wolpensinger,B., Lustig, A., Schatz, G., afolded substrate by the ATP-dependent lon protease. J. Biol. Chem. 280: Engel,A., and Suzuki, C.K. 1999. Mitochondrial Lon of Saccharomyces 25103–25110. cerevisiae is aring-shaped protease with seven flexible subunits. Proc. Natl. Ovchinnikova, T.V.,Martynova, N.Yu., Potapenko, N.A., Vasilyeva, O.V., Acad. Sci. 96: 6787–6790. Golikova, N.Yu., Ginodman, L.M., and Rotanova, T.V. 1998. Study of heat Starkova, N.N., Koroleva, E.P., Rumsh, L.D., Ginodman, L.M., and shock bacterial proteins: Limited proteolysis of ATP-dependent protease Rotanova, T.V. 1998. Mutations in the proteolytic domainof Escherichia Lon from. E. coli. Biomed. Tekhnol. 8: 62–65. coli protease Lon impair the ATPase activity of the enzyme. FEBS Lett. Paetzel, M., Dalbey,R.E., and Strynadka, N.C. 2002. Crystal structure of 422: 218–220. abacterial signal peptidaseapoenzyme:Implicationsfor signal peptide Stock, D., Gibbons,C., Arechaga, I.,Leslie, A.G.,and Walker, J.E. 2000. The binding and the Ser-Lys dyad mechanism. J. Biol. Chem. 277: 9512– rotary mechanismofATP synthase. Curr. Opin. Struct. Biol. 10: 672– 9519. 679. Park, S.C., Jia, B., Yang, J.K., Van, D.L., Shao, Y.G., Han, S.W., Jeon, Y.J., Tsirulnikov,K.B., Melnikov,E.E., and Rotanova, T.V. 2003. Proteolysis Chung, C.H., and Cheong, G.W.2006. Oligomeric structure of the ATP- coupledwith ATP. Regulation of activity of proteolytic centers of dependent protease La (Lon) of Escherichia coli. Mol. Cells 21: 129–134. Escherichia coli lon protease. Bioorg. Khim. 29: 486–494. Patterson, J., Vineyard, D., Thomas-Wohlever,J., Behshad, R., Burke, M., and van Dijl, J.M., Kutejova, E., Suda, K., Perecko, D., Schatz, G., and Lee, I. 2004. Correlation of an adenine-specific conformational change Suzuki, C.K. 1998. The ATPase and protease domains of yeast mitochon- with the ATP-dependent peptidase activity of Escherichia coli Lon. Bio- drial Lon: Roles in proteolysis and respiration-dependent growth. Proc. chemistry 43: 7432–7442. Natl. Acad. Sci. 95: 10584–10589. Peat, T.S., Frank, E.G., McDonald, J.P., Levine, A.S., Woodgate,R., and VanDyck, L., Pearce, D.A., and Sherman, F. 1994. PIM1 encodes amitochon- Hendrickson, W.A. 1996. Structure of the UmuD9 protein and its regulation drial ATP-dependent protease that is required for mitochondrial function in response to DNAdamage. Nature 380: 727–730. in the yeast . J. Biol. Chem. 269: 238–242.

www.proteinscience.org 1827 JOBNAME: PROSCI 15#8 2006 PAGE: 14 OUTPUT: Tuesday July 11 00:19:17 2006 csh/PROSCI/118158/ps0520693

Downloaded from www.proteinscience.org on August 3, 2006

Rotanova et al.

Vasilyeva, O.V., Kolygo, K.B., Leonova, Y.F.,Potapenko, N.A., and Waxman, L. and Goldberg, A.L. 1982. Protease La from Escherichia coli Ovchinnikova, T.V. 2002. Domain structure and ATP-induced conforma- hydrolyzes ATPand proteins in alinked fashion. Proc. Natl.Acad. Sci. 79: tional changesin Escherichia coli protease Lonrevealed by limited 4883–4887. proteolysis and autolysis. FEBS Lett. 526: 66–70. Weber-Ban, E.U., Reid, B.G., Miranker,A.D., and Horwich, A.L. 1999. Global Vineyard, D., Patterson-Ward, J., Berdis, A.J., and Lee, I. 2005. Monitoring the unfolding of asubstrate proteinbythe Hsp100 chaperone ClpA. Nature timing of ATPhydrolysis with activation of peptide cleavage in Escherichia 401: 90–93. coli Lon by transient kinetics. Biochemistry 44: 1671–1682. Whitby,F.G., Masters, E.I., Kramer,L., Knowlton, J.R., Yao, Y.,Wang, C.C., von Janowsky,B., Knapp, K., Major,T., Krayl, M., Guiard, B., and V oos, W. and Hill, C.P.2000. Structural basis for the activation of 20S proteasomes 2005. Structural properties of substrate proteins determine their proteolysis by 11S regulators. Nature 408: 115–120. by the mitochondrial AAA+ protease Pim1. Biol. Chem. 386: 1307–1317. Wickner,S., Maurizi, M.R., and Gottesman, S. 1999. Posttranslational quality Wang, J. 2004. Nucleotide-dependent domain motions within rings of the control: Folding, refolding, and degrading proteins. Science 286: 1888– RecA/AAA(+)superfamily. J. Struct. Biol. 148: 259–267. 1893. Wang, J., Hartling,J.A., andFlanagan, J.M. 1 997. Thestructure o fClpPat2.3 A ˚ Xia, D., Esser,L., Singh, S.K., Guo, F.,and Maurizi, M.R. 2004. Crystallo- resolution suggests amodel forATP-dependentproteolysis. Cell 91: 447–456. graphic investigation of peptide binding sites in the N-domain of the ClpA Wang, J., Song, J.J., Franklin, M.C., Kamtekar,S., Im, Y.J., Rho, S.H., chaperone. J. Struct. Biol. 146: 166–179. Seong, I.S., Lee, C.S., Chung, C.H., and Eom, S.H. 2001. Crystal structures Zhang, X., Shaw,A., Bates, P.A., Newman, R.H., Gowen, B., Orlova, E., of the HslVU peptidase-ATPase complex reveal an ATP-dependent pro- Gorman, M.A., Kondo, H., Dokurno, P.,Lally,J., et al. 2000. Structure of teolysis mechanism. Structure 9: 177–184. the AAA ATPase p97. Mol. Cell 6: 1473–1484.

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