Báo cáo khoa học: The Thermoplasma acidophilum Lon protease has a Ser-Lys dyad active site pot

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PRIORITY PAPERTheThermoplasma acidophilumLon protease has a Ser-Lys dyadactive siteHenrike Besche and Peter ZwicklDepartment of Structural Biology, Max-Planck-Institute of Biochemistry, Martinsried, GermanyA g ene with significant s imilarity to bacterial Lon proteaseswas identified during the s equencing o f the genome of thethermoacidophilic archaeon Thermoplasma acidophilum.Protein sequence comparison revealed t hat ThermoplasmaLon protease ( TaLon) is more similar t o t he LonB proteasesrestricted to Gram-positive bacteria than to the widely dis-tributed bacterial L onA. However, th e active site residues ofthe protease and ATPase domain a re highly conserved i n allLon proteases. U sing site-directed mutagenesis we s how herethat TaLon and EcLon, and probably all other L on pro-teases, contain a Ser-Lys dyad activ e site. The Ta Lon activesite mutants were fully assembled and, similar to TaLonwild-type, displayed an apparent molar mass1of 430 kDaupon gelfiltration. This would be consistent with a hexamericcomplex and indeed electron micrographs of TaLonrevealed ring-shaped p articles, although of unknown sym-metry. Comparison of the ATPase activity o f Lon wild-typefrom Therm oplasma or Escherichia coli with respective pro-tease a ctive s ite mutants revealed differences in KmandV values. T his suggests th at in the course of protein d egra-dation by wild-type Lon the protease domain might influ-ence the activity of the ATPase domain.Keywords:AAA+protease; archaea; Lon(La) endopepti-dase; Lon (La) protease; Ser-Lys dyad.Endopeptidase La (EC was the first ATP-dependent proteo lytic enzyme to be identified [1,2]. Laterprotease La was found to be the product of theEscherichia coli lon gene2[3,4] and is now mainly calledLon p eptidase or protease. T his c an be seen by therespective entry and listed references in the MEROPSpeptidase database: peptidase family S16 (lon proteasefamily) (http://merops.sanger.ac.uk/famcards/summary/s16.htm) [5].The L on protease domain is ubiquitously distr ibutedand m ostly fused to different ATPase domains [6].However, in the genomes of some Bacteria and A rchaea,standalone Lon protease domains are present, but nothingis known about their biological function. In contrast,plenty of knowledge has accrued about bacterial andmitochondrial Lon pr oteases [7–10], where t he Lon domainis linked t o an N -terminal AAA+domain which, i n turn, isextended N-terminally by a Lon N-terminal (LAN)domain [6]. Certain bacteria contain a second Lonprotease, called LonB, which lacks the LAN domain [6]and is assumed to be soluble [11]. Most Archaea contain aLonB homologue, i.e. lacking the LAN domain, whichcontains two transmembrane-spanning regions and w asshown to b e membrane-bound [12,13].It has been known for a l ong time that Lon proteaseshave an active site serine residue [14], but despite extensivemutagenesis the residual catalytic residues r emained e lusive[15]. Ultimately, mutagenesis studies of a viral noncanon-ical Lon protease l acking the ATPase domain revealed thecatalytic Ser-Lys dyad ( S-K dyad) for Lon p roteases [16].We set out to mutate the conserved serine and lysineresidues in Thermoplasma acidophilum Lon protease(TaLon) to generalize the S-K dyad for ATP-dependentmembrane-bound Lon proteases. In the course o f thiswork an independent report confirmed the S-K dyad forthe E. coli Lon protease (EcLon), but without studyingmutual regulation of the ATPase a nd protease domain [ 17].Based on mutagenesis studies it was p roposed that theATPase domain of EcLon regulates the protease in aunidirectional manner, i.e. the mutational inactivatedprotease domain did not influence t he ATPase activity[18]. We investigated this aspect in more detail for TaLonand Ec Lon b y detailed a nalysis o f p rotease active s itemutants.Materials and methodsSequence alignmentsThe a ctive-site regions of Lon protease protein sequenceswere aligned withCLUSTAL X[19] on Macintosh PPC.Correspondence to P. Zwickl, Max-Planck-Institute of Biochemistry,Department of Structural Biology, Am Klopferspitz 18,82152 Martinsried, Germany. Fax: +49 89 85782641,Tel.: +49 89 85782647, E-mail: zwickl@biochem.mpg.deAbbreviations: AAA+, ATPase associated with various cellularactivities; DDM, dodecyl-b-D-maltopyranoside; EcFtsH, Escherichiacoli FtsH; EcLon, Escherichia c o li Lon; LAN, LonN-terminal; Lonwt,Lon wild-type; S-K, Ser-Lys; TaLon, Thermoplasma acidophilum Lon;TaLonwt, Thermoplasma acidophilum Lon wild-type.Enzyme: e ndopeptidase L a (EC 19 August 2 0 04, revised 1 O cto ber 2004,accepted 6 October 2004)Eur. J. Biochem. 271, 4361–4365 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04421.xGeneration of active site mutantsSite-directed mutagenesis was performed using the Q uick-ChangeÒ Kit (Stratagene, La Jolla, CA, USA)3.pET22b(+)-TaLon-His6(S525A and K568A) and Lonwild-type (pLonwt) (K722A) served a s PCR t emplates. Therespective primers are listed with the mutated codonsunderlined: S525A, 5¢-CGAGGGAGTTGAAGGAGACGCGGCCAGCGTATCAATAGCC-3¢ (sense), 5¢-GGCTATTGATACGCTGGCCGCGTCTCCTTCAACTCCCTCG-3¢ (antisense); K568A, 5 ¢-CCGGTTGGCGGCGTAACCGCAGCGGTTGAGGCAGCTATAGAAGC-3¢ (sense),5¢-GCTTCTATAGCTGCCTCAACCGCTGCGGTTACGCCGCCAACCGG-3¢ (antisense); K7 22A, 5¢-GCCGATGGTGGTTTGAAAGAAGCCCTCCTGGCAGCGCATCGCG -3¢ (sense), 5¢-CGCGATGCGCTGCCAGGAGGGCTTCTTTCAAACCACCGATCGGC-3¢ (a ntisense).DNA sequencing ( MWG, E bersberg, Germany)4of thefull-length gene che cked all generated plasmids.Expression, purification and enzymic characterizationWild-type a nd muta nt Ta Lon p roteases were produced, andthe hydrolysis of fluorigenic peptides, FITC-labelled caseinand ATP were assayed as described [13]. R. G lockshuber(ETH Zu¨rich, Switzerland) kindly provided the plasmidspLonwt and pLonS679A [20]. Cells of E. coli BL21(DE3)(Novagen, M adison, WI, USA)5harbouring p Lon (-wt,-S679A or -K722A) were grown at 37 °Cin6LLuria–Bertani m edium containing 100 lgÆmL)1ampicillin. A t anattenuance6of 0.6 (600 nm) isopropyl thio-b-D-galactosidewas a dded to a final concentration o f 1 mM.Afterfurtherincubation for 4 h the cells were harvested b y centrifugation(4000 g,10min4°C),washedwith50mMTris (pH 7.5)and s tored until purification at )80 °C. Purification of theEcLon variants was performed as described [20].ResultsDomain organization of Lon proteasesDuring the sequencing of the T. ac idophilum genome anopen reading frame (ORF 1081) was identified, whichshowed significant sequence similarity to Lon proteases [21](Fig. 1 A,B). TaLon encompasses an N-terminal ATPaseassociated with various cellular a ctivities ( AAA+domain)and a C-terminal protease domain, but lacks the N-terminala-helical domain inherent in most bacterial and eukaryoticLon homologues (Fig. 1C). However, the AAA+domainof TaLon contains an insert of approximately 9 0 a mino acidresidues between the Walker A and Walker B ATPasesignatures [22], which is not present in any bacterial oreukaryotic Lon sequence. The 90-residue insert is found inall archaeal L on homologues and is predicted t o containtwo consecutive transmembrane h elices, suggesting t hatarchaeal Lon proteases are membrane a ssociated [ 21].Gram-positive bacteria, such as Bacilli and Clostridia,contain a second Lon protease, called LonB which, like t hearchaeal Lon homologues, does not contain an N-terminala-helical domain ( Fig. 1C) . In contrast with archaeal Lonproteases the b acterial LonB also lacks the 90-residue insertharbouring the predicted t ransmembrane region and istherefore probably a soluble protease, although this h as notbeen addressed experimentally [11].Notably, the archaeon Methanosarcina mazei has t wo longenes, an ar chaeal-type lon gene containing two transmem-brane helices and a bacterial-type lon gene including anN-terminal domain (Fig. 1). The M. maze i archaeal-typelon gene was not included in a phylogenetic analysis [23]although both genes are a lso p resent in the closely relatedspecies Methanosarcina acetivorans and M. barkeri. Simi-larly, the M. mazei genome contains both the comp letegroup I and group II chaperonin systems, i.e. the bacterialGroEL/GroES and the archaeal thermosome/prefoldin[24]. In general, approximately 31% of the ORFs in thegenomes of M. mazei and its close relatives share t he highestsimilarity with bacterial genes, which is most probably aresult of horizontal gene transfer [23]. In addition to thebacterial and archaeal lon genes the genome of M. mazeicontains a noncanonical lon gene (ORF Mm1931), enco-ding for a Lon protease lacking the ATPase domain.Noncanonical Lon proteases are also found in bacteria andviruses (Fig. 1B,C). A separate study failed to detect theATP-binding region in the archaeal Lon proteases insequence c omparisons with bacterial homologues andclassified the Pyrococcus Lon homologues as ATP-inde-pendent proteases [25]. This is easily explained by t he factFig. 1. Sequence a lignment o f s elected r egions of distinct Lon proteases.(A) Alignment of the Walker A an d W alker B motifs of the AAA+ATPase-domain. Identical residues are marked with #, conservedresidues with +. (B) A lignment of the S-K dyad of th e p rotease do-main. Labelling as described in ( A). Essential residues o f TaLon pro-tease activity are indicated below. (C) Schematic representation of thedomain organization of the prote ins aligned in (A) and ( B) . Bs, Bacillussubtilis;Ec,Escherichia coli;IBDVP2,infectiousbursal disease virusstrain P2; M m, Methanosarcina mazei;Ta,Thermoplasma ac idophilum;Tk, Th ermo coccus kodakarensis. The UniProt a ccession numbers ofthe aligned proteins are: Bs-LonA, P37945; Bs-LonB, P42425;Bs-YlbL, O34470; Ec-Lon, P08177; I BDVP2-VP4, Q82628; Mm-1913,Q8PVP9; Mm-bLon, Q 8PSG1; Mm-Lon, Q8Q0K8; Ta-Lon,Q9HJ89; Tk-Lon, Q8NKS6.4362 H. Besche and P. Zwickl (Eur. J. Biochem. 271) Ó FEBS 2004that archaeal Lon p roteases contain a 90 -re sidue insertbetween the W alker A and Walker B motifs as mentionedabove, which impairs alignment of the bacterial with t hearchaeal ATPase domain. After removing the 90-residueinsert the bacterial and archaeal Walker A and W alker Bmotifs can b e aligned ( Fig. 1A). Sequence alignment o f theproteolytic core domain s hows that the active site S-K dyadis conserved in archaeal L on, in bacterial LonA and LonB,and in noncanonical Lon proteases ( Fig. 1B). In summary,the Lon protease domain i s an e volutionarily ancientdomain, wh ich i s ubiquitously distributed and c an be fusedto various other domains [6].TaLon forms ring-shaped complexesOverexpression in E. coli leads to m embrane insertion ofthe TaLon protease. The isolated membranes were solu-bilized with detergent, and r ecombinant TaLon waspurified by Ni2+-nitrilotriacetic a cid a ffinity chromatogra-phy [13]. Subsequent size exclusion chromatographyrevealed an apparent molar mass of 510 kDa. Thetransmembrane domain of TaLon is surrounded by adodecyl-b-D-maltopyranoside (DDM) micelle with a massof  70 kDa. Subtraction of the DDM micelle suggeststhat six TaLon mo no mers ( 72 kDa) ass emb le in to ahexameric c omplex (Fig. 2A). C onsistently, electron micro-scopic analysis revealed r ing-shaped particles of yetunknown symmetry ( Fig. 2B). The isolated p roteolyticdomain of EcLon w as crystallised as a hexameric complex[26]. In contrast, a structural analysis of the yeastmitochondrial Lon protease by analytical ultracentrifuga-tion and electron m icroscopy revealed heptameric ring-shaped complexes [27]. The stoichiometry of the TaLoncomplex remains to be determined.S-K dyad active siteLon from E. coli has been known for a l ong time to be aserine protease [14,20], but only lately was an active sitelysine residue7identified [16,17]. These two catalytic residuesare conserved among all a rchaeal and bacterial Lonhomologues and form an S-K d yad. Very recently, thecrystal structure of the hexameric E. coli Lon proteasedomain has been solved and revealed a unique fold notobserved in other S-K dyad peptidases [26]. Only thecatalytic core containing the active s ite residues is structur-ally conserved between distinct S-K dyad peptidases [26].To establish that the Therm oplasma me mbrane-bound Lonprotease has the same active site, t he two conserved residueswere individually muta ted to alanine (S52 5A and K568A).As with TaLonwt, the S -K mutants were purified ashexamers (data not shown) sustaining substantial ATPaseactivity (Table 1), but no peptidase activity could bedetected (Fig. 3 A). Consequently, TaLonS525A andTaLonK568A showed neither ATP-dependent nor ATP-independent proteolytic activity (Fig. 3B).ATPase activity of protease active site mutantsKinetic analysis of TaLon and its protease active sitemutants r evealed t hat t he specific ATPase a ctivity o fFig. 2. Molar mass and electron microscopy of TaLon. (A) TaLo n(30 mg) was s eparated on Supe rdex 200 HiLoad 26/60 column (25 mMMes pH 6.2, 300 mMNaCl, 5 mMMgCl2,0.5mMDDM) anddetected by UV280. M olar mass of marker pro teins an d their respectiveelution volume s are in dicated. G el filtration r evea led an a pparen tmolar mass o f 510 kDa corresponding to a hexameric complex a ftersubtraction of 70 kDa for the DDM micelle. (B) Electron micrographof negatively stained (2% uranylacetate) TaLo n from the Superdex200 peak fraction, recorded with a Philips C M 200 FEG transmissionelectron microscope at 16 0 kV.Table 1. ATPase ac tivity of TaLon and EcLon.Protease Km(mM) V (PiÆmin)1Ælg)1)TaLonwt 0.196 ± 0.004 0.631 ± 0.003TaLonS525A 0.176 ± 0.014 0.561 ± 0.012TaLonK568A 0.111 ± 0.003 0.477 ± 0.003EcLonwt 0.201 ± 0.004 0.554 ± 0.012EcLonS679A 0.140 ± 0.014 0.273 ± 0.009EcLonK722A 0.189 ± 0.011 0.782 ± 0.015Ó FEBS 2004 T. acidophilum Lon is a Ser-Lys dyad protease (Eur. J. Biochem. 271) 4363TaLonK568A was 25% lower and the affinity for ATPwas increased by 43% when compared with wild-typeTaLon (Table 1). The Kmof TaLonS525A remainedunaffected and the specific ATPase activity was onlyslightly reduced (10%; T able 1). Thus the mutation o f thecatalytic protease residues, especially of Lys568 seems toenhance ATP binding and concomitantly slow down ATPhydrolysis. In order to generalize this observation thecorresponding E. coli lysine mutant was generated (EcLon-K722A) and purified along with the active site serinemutant (EcLonS679A). B oth m utants were proteolyticallyinactive (data not shown). I n accordance with our modelthat the protease domain might have a r egulative influenceon the protease domain, both mutants were affected intheir ATPase activity i n comparison with the wild-typeenzyme. The EcLon Ser679 mutant showed only h alf thewild-type ATPase activity but a 30% higher affinity forATP, while the lysine mutant w as stimulated with resp ectto ATP hydrolysis (40%) and hardly affected in its affinityfor ATP (Table 1).DiscussionThe Lon protease is ubiquitously distributed and was foundto exist as a standalone protein as well as fused to a AAA+ATPase-domain (Fig. 1C ) [6]. Whereas the b acterial Lonprotease and its homologues i n eukaryal organelles aresoluble, the archaeal c ounterpart is membrane-attached[13]. Bacterial LonB proteases, like archaeal Lon, lack theLAN domain p resent in bacterial L onA pro teases, but likebacterial LonA, also miss the membrane anchor foun d inarchaeal Lon proteases. Taken together the Lon proteasedomain is h ighly versatile and can function in differentcontexts, i.e. standalone or fused t o an ATPase, and s olubleor membrane-bound.Solubilized TaLon has a m olar mass of 430 kDacorresponding to a hexameric complex. T he s ame o ligome-rization state w as revealed r ecently in t he crys tal structure ofthe hexameric EcLon protease domain [26], whereaselectron microscopy of yeast m itochondrial Lon proteaeseshowed a heptameric complex [27].Using site-directed mutagenesis we established t he S-Kdyad for the membrane-bound Ta Lon and confirmed it f orthe soluble EcLon. Subsequently, we characterized theTaLon and EcLon protease-deficient mutants lacking thecatalytic serine o r lysine residue. K inetic analysis andcomparison of their r espective ATPase activity revealed apotential regulation of ATPase hydrolysis depending onthe p roteolytic cycle. Though the unidirectional regulationof the peptidase activity by the ATPase has been describedfor the EcLon protease several years ago [18], thereciprocal regulatio n of the ATPase b y the proteaseremained unrecognized, altho ugh two reports describedimpaired ATP hydrolysis of FtsH m utants lacking p roteaseactivity. Karata et al . [28] reported that mutation of thezinc-binding residue His421 in Escherichia coli FtsH(EcFtsH) completely abolished protease activity an dreduced the ATPase activity to 23% of wild-type FtsH.In this case, it c an be claimed that the prevention of zincion b inding might l ead to structural perturbations thatreduce the ATPase activity. However, i n two independentstudies mutations of the catalytic aspartate in the conservedHEAGH motif of EcFtsH [29] and Bacillus subtilis FtsH[30] were generated. The conserved aspartate residueactivates a w ater molecule for the nucleo philic attack butdoes not affect zinc ion binding. Again these mutant Fts Hproteins were reported to lack proteolytic activity andshowed a 20% reduced ATPase activity. Taken togetherwith our observation that mutation of the Ta Lon andEcLon protease active site residues dec reases the ATPaseactivity, we propose that bidirectional crosstalk betweenthe ATPase a nd peptidase domains is necessary forcontrolled protein degradation.Fig. 3. Pr oteoly tic activity of TaLonwtandtheactivesitemutantsTaLonS525A and TaLonK568A. (A) TaLon (46 nM)andThermo-plasma acidophilum p roline iminopeptidase (1.83 lM)wasincubatedwith 100 lMsuccinyl-LLVY-7-amido-4-methylcoumarin in assaybuffer ( 50 mMMes pH 6.2, 20 mMMgCl2,0.5mMDDM) at 60 °C.TaLonwt and the ATPase d eficient mu tant Ta LonK63A (compare[13]) served as control; the wild -type activity was set equal to o ne . (B)TaLon (116 nM)wasincubatedwith5lMfluorescein isothiocyanate-casein i n the assay buffer with or without 2 mMATP a t 60 °C. For theS-K dyad m utants n o diffe renc e w as obse rved in the presence orabsence o f ATP.4364 H. Besche and P. Zwickl (Eur. J. Biochem. 271) Ó FEBS 2004AcknowledgementsWe tha nk Ulf Klein (MPI Martinsried) f or assistance, Oana Mihalache(MPI Martinsried) for e lectron microscopy, Erik Roth (MPI Martins-ried) for calibration of the Sup erdex 200 column and Rudi Glockshuber(ETH Zu¨rich) for providing EcLon expression plasmids. We areindebted to Wolfgang Baumeister (MPI Martinsried) for g enerous a ndcontinuous support. Fin ally, we want to thank the referees for theirvaluable suggestions. This work was supported by a grant from theDFG t o Peter Z wickl (Z w58/3-2).References1. Swamy, K.H. & Goldberg, A.L. (1981) E. coli conta ins e i ghtsoluble proteolytic activities, o ne being ATP dependent. Nature292, 652–654.2. Chung, C.H. & Goldberg, A.L. (2004) Endopeptidase La. InHandbook of Proteolytic E nyzmes (Barret,A.J.,Rawlings,N.D.&Woessner, J.F., eds), pp. 1998–2002. Elsevier Academic Press,London, UK.3. Chung, C.H. & Goldberg, A.L. 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Biophys. 380, 1 03–107.30. Kotschwar,M.,Harfst,E.,Ohanjan,T.&Schumann,W.(2004)Construction and analyses of mutant ftsH alleles of Bacillussubtilis involving the ATPase- a nd Zn-binding domains. Curr.Microbiol. 49, 1 80–185.Ó FEBS 2004 T. acidophilum Lon is a Ser-Lys dyad protease (Eur. J. Biochem. 271) 4365 . wild-type Lon the protease domain might influ- ence the activity of the ATPase domain. Keywords:AAA + protease; archaea; Lon( La) endopepti- dase; Lon (La) protease; Ser-Lys dyad. Endopeptidase La (EC. [21] (Fig. 1 A, B). TaLon encompasses an N-terminal ATPase associated with various cellular a ctivities ( AAA + domain) and a C-terminal protease domain, but lacks the N-terminal a- helical domain inherent. acterial Lon protease and its homologues i n eukaryal organelles are soluble, the archaeal c ounterpart is membrane-attached [13]. Bacterial LonB proteases, like archaeal Lon, lack the LAN domain
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