Biochimica et Biophysica Acta 1784 (2008) 727–735

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Review

Functional mechanics of the ATP-dependent Lon protease- lessons from endogenousprotein and synthetic peptide substrates

Irene Lee a,⁎, Carolyn K. Suzuki b,⁎a Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-7078, USAb Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, MSB E661, Medical Science Building, 185 South Orange Avenue, Newark,NJ 07101-1709, USA

a r t i c l e i n f o

⁎ Corresponding authors. I. Lee is to be contacted at tetel.: +1 973 972 1555.

E-mail addresses: IRENE.LEE@case.edu (I. Lee), SUZUKAbbreviations: ATP, adenosine triphosphate; ADP, ade

adenylyl 5-imidodiphosphate; Abz, anthranilamide; Bz,O-(N-methylanthraniloyl); Dansyl, 5-dimethylamino-fluorescein isothiocyanate; Abu, amninobutyric acid

1570-9639/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.bbapap.2008.02.010

a b s t r a c t

Article history:Received 31 December 2007Received in revised form 17 February 2008Accepted 20 February 2008Available online 5 March 2008

Lon, also knownas the protease La, is a homo-oligomeric ATP-dependent protease,which is highly conserved inarchaea, eubacteria and eukaryotic mitochondria and peroxisomes. Since its discovery, studies have shownthat Lon activity is essential for cellular homeostasis, mediating protein quality control and metabolicregulation. This article highlights the discoveriesmade over the past decade demonstrating that Lon selectivelydegrades abnormal as well as certain regulatory proteins and thus plays significant roles in maintainingbacterial and mitochondrial function and integrity. In addition, Lon is required in certain pathogenic bacteria,for rendering pathogenicity and host infectivity. Recent research endeavors have been directed towardelucidating the reaction mechanism of the Lon protease by different biochemical and structural biologicaltechniques. In this mini-review, the authors survey the diverse biological roles of Lon, and also place specialemphasis on recent findings that clarify the mechanistic aspects of the Lon reaction cycle.

© 2008 Elsevier B.V. All rights reserved.

Keywords:Oligomeric ATP-dependent proteasesProteinPeptideMechanism

1. Introduction

The ATP-dependent Lon (La) protease derives its name from thephenotype of Escherichia coli lon genemutants that form long undividedfilaments upon UV irradiation [1–3]. Since its discovery, studies haveshown that Lon is essential for cellular homeostasis by mediating thedegradation of abnormal and damaged polypeptides, as well as short-lived regulatoryproteins. Lon functions in the cytosol of prokaryotes andin mitochondria and peroxisomes of eukaryotes [2,4–8].

1.1. Structure of Lon

The Lon protease is highly conserved in archaea, eubacteria, andeukaryotes. The Lon protein sequence of E. coli (EcLon) shows 50%sequence similarity with that of the archaebacterium Thermoplasmaacidophilum (TaLon), and 25% similarity with mitochondrial Lon fromthe budding yeast Saccharomyces cerevisiae (ScLon) and Homo sapiens(HsLon). The Lon holoenzyme is a homo-oligomer, in which eachsubunit carries a domain for ATP hydrolysis and proteolysis (Fig. 1);the greatest degree of sequence conservation surrounds these catalytic

l.: +1 216 368 6001. C.K. Suzuki,

ICK@umdnj.edu (C.K. Suzuki).nosine diphosphate; AMPPNP,benzoic acid; MANT, 2'-(or 3')1-naphthalenesulfonyl; FITC,

l rights reserved.

sites. The domain structure of Lon consists of an amino-terminal (N)domain that has been implicated in protein substrate binding andoligomerization [10], an ATPase (A) domain, a Substrate Sensor andDiscriminatory domain (SSD) that may also be involved in substratebinding, and a carboxyl-terminal (P) domain containing the Ser-Lys dyadat the proteolytic active site [9].

Structural studies demonstrate that the Lon holoenzyme is a ring-shaped complex composed of identical subunits. Analytical ultracen-trifugation analyses show that purified Lon of Mycobacteriumsmegmatis (MsLon) is hexameric, and that oligomerization is depen-dent on Mg2+, but independent of ATP, and stimulated by unfoldedprotein. MsLon monomers are in reversible equilibrium with oligo-meric sub-complexes (e.g. dimers, trimers, tetramers) and completehexameric complexes [11]. Electron microscopy of EcLon reveals asingle hexameric ring-shaped structure with a central cavity [12]. LikeMsLon, the formation of EcLon hexamers is Mg2+-dependent and ATP-independent [12]. Cryoelectron microscopy and analytic ultracentri-fugation of ScLon demonstrates that the mitochondrial protease is asingle heptameric ring-shaped complex with flexible subunits [13].Maintenance of the ScLon holoenzyme does not require the additionof Mg2+ or ATP to the purified protease. In the absence of addednucleotide, two adjacent subunits within the ScLon complex are ex-tended resulting in an asymmetric ring. Upon addition of ATP or non-hydrolyzable analogs of ATP, the percentage of symmetric ring-shapedcomplexes is increased, suggesting that ATP binding induces con-formational changes in the ScLon holoenzyme.

Lon is structurally distinct from other soluble two-component ATP-dependent proteases such as HslUV and ClpAP in bacteria, ClpXP in

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bacteria and metazoans, and the 26S proteasome in eukaryotes. Thesetwo-component proteases consist of separate complexes that mediateATP hydrolysis and proteolysis. The ATPase component consists of sixidentical subunits-HslU, ClpA or ClpX that assemble with their corres-ponding protease components- HslV or ClpP, which consist of sevenidentical subunits. Despite differences in the overall architecture ofLon and these two-component proteases, Lon, HslU, ClpA and ClpX allbelong to the AAA+ family of proteins (ATPases Associated with avariety of cellular Activities), whose members are involved in DNAreplication, transcription, membrane fusion, and proteolysis [14–17].This family of proteins shares a conserved ATP-binding module of~200 amino acids that assembles into oligomeric rings. The AAA+

domain of Lon shows a significant degree of sequence homology withthe HslU, ClpA and ClpX, therefore it is speculated that the function ofATP binding and hydrolysis by these enzymes is mechanisticallysimilar.

Although the atomic structure of Lon has not been solved, X-raystructures of the N-domain, part of the AAA+ domain, and the P-domain have been independently determined [9,18–20] (see Fig. 1 fordomain organization). The P-domains of various bacterial Lons showhexameric ring-shaped structures. Although these P-domains assem-ble to form hexameric structures, they lack proteolytic activity. Site-directedmutagenesis reveals that the catalytic Ser–Lys dyad located inthe P-domain of Lon is essential for proteoytic but not ATPase activity[18,21,22], and that amino acids in addition to the Ser–Lys dyad mayparticipate in catalyzing peptide bond cleavage [18,22–24]. Interest-ingly, the ATPase activity of EcLon is affected by a variety of mutationsgenerated at the vicinity of the proteolytic site Ser 679 [22]. Further-more, mutation of the ATP-binding site abolishes both the ATPase andprotease activities of EcLon [21]. These results collectively suggest thatcommunication between the ATPase and the proteolytic sites is crucialto Lon catalysis (more detailed discussion is provided below). Inaddition, the catalytic activity of Lon is also dependent on inter-sub-unit interactions. Mixed oligomeric complexes composed of wild-typeEcLon and the inactive EcLon E614K mutant, results in an enzyma-tically inactive protein [25]. Currently, a high-resolution structureof eukaryotic Lon is unavailable, and the oligomeric state of mam-malian Lon has yet to be determined. Due to conservation in thesequences of the functional domains among prokaryotic and eukar-yotic Lon homologs, it is speculated that the architecture and under-lying mechanism of these enzymes will be similar.

1.2. Physiological functions of Lon

As a heat shock protein, one of themain functions of Lon is to act asa quality control protease. In bacteria, Lon mutants exhibit a defect inabnormal protein turnover, demonstrating that EcLon functions inprotein quality control. EcLon is also important for selectively degra-ding short-lived regulatory proteins, such as bacteriophage λ N pro-

Fig. 1. Domain organization for

tein, the SulA cell division regulator; the positive regulator of capsulesynthesis, Rcs; and the F factor addiction system protein CcdA (forreviews see [5,26,27]). In the bacterium Caulobacter crescentus, Lonselectively degrades the Cell-Cycle-Regulated DNA Methyltransferase(CcrM) thereby regulating the methylation of chromosomal DNA andcellular differentiation [28]. Pathogenic bacteria, such as Brucellaabortus and Salmonella enterica serovar Typhimurium (S. typhimurium),Lon-mediated proteolysis of the central transcription regulatory factorHilA, controls the correct timing for the expression of virulence genesnecessary for host invasion [29–31]. In Pseudomonas aeruginosa, theinduction of Lon has been proposed to contribute to the adaptiveresistance of the organism towards antibiotic treatment [32].

In eukaryotes, the absence or down-regulation of Lon results indiverse phenotypes. In S. cerevisiae, the absence of ScLon, results ina lack of ATP-dependent proteolysis in the mitochondrial matrix,accumulation of electron dense aggregates and large mtDNA deletions[33,34]. In yeast, ScLon is the only ATP-dependent protease within thematrix, by contrast to metazoans where the two-component ClpXPprotease is also present. Endogenous substrates of ScLon are generallymisfolded or unassembled subunits of electron transport chain com-plexes, ribosomal proteins and metabolic enzymes [35–38]. In humanlung fibroblasts, HsLon depletion for 4 days using anti-sense mor-pholino oligonucleotides results in defects in mitochondrial mem-brane potential, respiration and morphology, as well as apoptotic celldeath [39,40]. However, the effect of HsLon depletion may be celltype- or cell line-specific. The expression of an inducible short hairpinRNA leading to HsLon depletion in a colon adenocarcinoma cell linefor 14 days does not lead to cell death [7]. Even after RNAi knockdownfor ~3 weeks, these cells continue to survive, although they no longerproliferate (B. Lu and C.K. Suzuki, unpublished observations). Endo-genous substrates of mammalian Lon include subunits of cytochromec oxidase (COX), which is an electron transport chain complex [41,42];oxidized mitochondrial aconitase, which functions in the tricarboxylicacid cycle [43]; and the steroidogenic acute regulatory protein StAR,which mediates cholesterol transfer from the cytosol to the mito-chondrial inner membrane [44,45].

In addition to mediating ATP-dependent protein degradation,Lon also exhibits a chaperone-like function in mitochondria. In yeastand mammalian mitochondria, Lon has been found to promote theassembly of protein complexes independently of its protease activity[42,46]. In mammals, a stress response network between the endo-plasmic reticulum (ER), nucleus and mitochondrion has been shownto up-regulate the expression of Lon in rat and human cells, therebyproviding a cytoprotective function during ER stress and hypoxia [42].ER stress and hypoxia lead to the accumulation of abnormal proteinswithin the ER and defects in mitochondrial function. ER stress andhypoxia stimulate the transcriptional up-regulation of ER-specificstress proteins [7]. The up-regulation of HsLon upon ER stress orhypoxia has been proposed to increase the assembly of subunit 2 of

a subunit in Lon protease.

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cytochrome c oxidase (Cox2) into functional complexes and to par-tially restore mitochondrial function. In yeast and mammalianmitochondria, defects in COX assembly are suppressed upon over-expressing wild-type as well as protease inactive ScLon and HsLon[7,46]. By contrast, COX assembly defects are not suppressed by anATPase mutant of ScLon [46]. Furthermore, a ScLon mutant lackingboth ATPase and protease activity also fails to suppress COX assemblydefects, suggesting that an ATP-dependent chaperone-like activity ofScLon is required for promoting protein complex assembly [49]. Thus,mitochondrial Lon may have a chaperone-like function independentof its proteolytic activity.

Recent work shows that HsLon functions in the adaptive responseto hypoxia, by remodeling the COX holoenzyme [41]. During normo-xia, an isoform of the Cox4 subunit, Cox4-1, is a stable component ofthe fully assembled enzyme complex. However, during oxygen depri-vation, the hypoxia-inducible transcription factor HIF-1α activates theexpression of an alternate isoform, Cox4-2, as well as HsLon. Increasedlevels of HsLon are required for the degradation of Cox4-1, therebypermitting Cox4-2 assembly into the COX holoenzyme. It is hypothe-sized that subunit switching produces Cox4-1 or Cox4-2 containingcomplexes that are each optimized for transferring electrons undernormoxic or hypoxic conditions, respectively. It is conceivable thatHsLon mediates not only the degradation of Cox4-1 but also theassembly of Cox4-2 into the holoenzyme complex.

2. Lon as a DNA binding protein

2.1. DNA binding by EcLon in vitro

Early studies identified that purified EcLon (also referred to CapR)as a DNA binding protein [47]. EcLon binds to double-stranded DNA(dsDNA) with a relative higher affinity than to single-stranded DNA(ssDNA) [48,49]. Several in vitro studies show that bacterial Lon inter-acts non-specifically with DNA plasmids, calf thymus and salmonsperm DNA [48,50,51], whereas one study demonstrates sequence-specific EcLon binding to a 35 bp element referred to as pets [49].However, in a study conducted by a different group, the 35 bp petselement does not compete with the non-specific DNA binding ofEcLon even at an 100-fold molar excess [51]. The lack of sequence-specific DNA binding by EcLon is also suggested by the analysis ofchromosomal DNA fragments co-purified with EcLon, which shows noapparent sequence similarities [51]. It remains unclear whether thereis a single conserved site within Lon proteins that mediates DNAbinding. One study using BtLon shows that the SSD sub-region of theA-domain is sufficient for DNA binding [10], whereas experimentswith EcLon show that the A-domain lacking the SSD sub-region bindsDNA [51]. Different observations have been reported for the relation-ship between DNA binding and the enzymatic activities of bacterialLon [48,50–52]. One study showed that large DNA molecules such asplasmid, bacteriophage and calf thymus DNA stimulate the ATPaseand protease activities of Lon [48]. By contrast, another study obser-ved that DNA stimulated ATP hydrolysis but inhibited proteolysis [50].In summary, the above observations show that for prokaryotic Lon,the sequence-specificity of dsDNA binding and the relationship bet-ween DNA binding, ATP hydrolysis and proteolysis remain unclear. AsEcLon degrades several proteins involved in DNA methylation andRNA transcription [28,53,54], it is conceivable that non-specific orspecific DNA binding may function to localize EcLon to the bacterialchromosome.

2.2. DNA binding by HsLon in vitro

Nucleic acid binding by the Lon protease is conserved from bacte-ria to mammalian mitochondria. Unlike bacterial Lon, HsLon bindssingle-stranded but not double-stranded DNA and RNA oligonucleo-tides [55–57]. In vitro, HsLon specifically binds to G-rich ssDNA oligo-

nucleotides having consecutive guanine residues. Protein substratestimulates DNA binding by HsLon whereas ATP-binding inhibits thisinteraction [56]. Interaction of a protein substrate with HsLon maylead to the release of bound ATP or ADP, resulting in a nucleotide-freeor -depleted form of the enzyme that binds with higher affinity toDNA. ssDNA, however, has no effect on ATP hydrolysis or proteolysis.Recent biochemical and biophysical experiments demonstrate thatthe binding affinity of HsLon for single-stranded G-rich mtDNAsequences is determined by the propensity of these sequences to formparallel G-quadruplexes, which are high-order structures [58]. Whe-ther single-stranded mtDNA forms G-quadruplexes in vivo and howLon binding to these higher ordered structures function in mtDNAintegrity has yet to be determined.

2.3. DNA binding by HsLon in cultured cells

Human mtDNA consists of a heavy- and light- strand; the heavystrand is distinguished by a higher percentage of guanine and thymineresidues resulting in its greater mass. The small circular human mito-chondrial genome (~17 kb) encodes thirteen proteins involved inoxidative phosphorylation, two ribosomal RNAs (rRNAs) and twenty-two transfer RNAs (tRNAs). The GT-rich heavy strand encodes all ofthese proteins except one, as well as both rRNAs. Human mtDNAcontains a single non-coding region referred to as the control region(CR) that carries the light- and heavy- strand promoters (LSP andHSP, respectively), which are required for mtDNA replication andtranscription.

Studies performed in cultured cells show that HsLon binds prefe-rentially to CR, in addition to several other regions of the mitochon-drial genome [59]. Lon binds with the highest affinity to a single-stranded G-rich sequence overlapping LSP on the heavy strand.Analysis of themtDNA sequences bound by Lon in living cells reveals aG-rich consensus sequence. Biochemical and biophysical studies showthat Lon specifically binds to ssDNA sequences that have a propensityfor forming G-quartets (also referred to as G-quadruplexes or tetra-plexes) [58]. Bioinformatic analysis shows a good correlation betweenregions of the mitochondrial genome that are bound by Lon in cultu-red cells and regions that are predicted to form G-quartets [59].

2.4. Potential physiologlcal functions of DNA binding by EcLon and HsLon

Although the physiological function and importance of DNAbinding by Lon remains unclear, results from bacterial and mamma-lian systems provide insights. In E. coli, the adaptation to amino acidstarvation results in a dramatic increase in inorganic polyphosphate(polyP), which directly stimulates EcLon-mediated proteolysis of freeribosomal proteins and thereby down-regulates translation [60,61].PolyP competitively blocks DNA binding by EcLon in vitro and in vivo,suggesting that polyP-EcLon is released from DNA as an activatedcomplex, which degrades free ribosomal proteins. It has been spe-culated that EcLon may bind to chromosomal DNA adjacent to sitesbound by regulatory proteins, and may thus control the turnover ofsuch proteins. In a similar manner in mammalian cells, the prefe-rential binding of HsLon to the control region for mtDNA replicationand transcription may function to recruit the protease to a site whereit selectively degrades protein components of the replication andtranscription machinery. HsLon interacts with the mtDNA polymeraseγA subunit and with the mtDNA helicase Twinkle [62], which arecomponents of mitochondrial nucleoids that are considered to becrucial for the segregation and inheritance of mtDNA. It is conceivablethat HsLon mediates the proteolytic remodeling of mitochondrialnucleoids and that it also has a direct role in mtDNA metabolism andintegrity. Results show that cellular levels of HsLon influence thesensitivity of mtDNA to oxidative mtDNA damage [59]. In control cellswith normal levels of HsLon, oxidative stress leads to an increasedfrequency of mtDNA lesions. By contrast, HsLon-depleted cells show

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little if any mtDNA damage. These findings suggest that damage ispermitted when HsLon is present and prevented when HsLon isabsent. When HsLon is present and bound to mtDNA, it may facilitateoxidative mtDNA damage by stabilizing ssDNA or blocking lesionrepair. Conversely, when HsLon levels are reduced, single-strandedmtDNA is perhaps less vulnerable to DNA damage andmore efficientlyrepaired. HsLon depletion may also lead to higher steady-state levelsof substrate proteins that function as antioxidants or repair proteins.Further experiments are required to determine the role of HsLon inthe maintenance of mtDNA integrity.

3. Modulation of Lon protease activity in vivo

Lon-mediated protein turnover in vivo is likely modulated or regu-lated by factors that affect either the enzymatic activity of the proteaseand/or the conformational state of protein substrates. As Lon is astress-induced protein that functions in cellular homeostasis andsurvival, it is not surprising that the activity of this protease is sub-jected to modulation by cellular metabolites such as polyP, [ADP]/[ATP] ratios, and protein substrate concentrations. Unfolded proteinsubstrates activate both the peptidase and ATPase activities of Lon[2,63]. For example, casein stimulates both the ATPase and peptidaseactivity of EcLon by interacting with an allosteric protein-binding siteon EcLon [64–66]. This finding may have functional implication in theactivation of mitochondrial Lon observed during oxidative stress [43].The production of oxidatively modified proteins may cause proteinunfolding. The unfolded oxidatively modified proteins then becomeallosteric activators and substrates of Lon. In addition, the bacter-iophage T4 PinA protein specifically inhibits EcLon but not ClpAP orHslUV (ClpYQ) [64]. PinA does not interact with the ATP-binding site,the proteolytic active site or the allosteric protein-binding site of EcLon.Rather, it appears to interact at a novel regulatory or enzymatic siteinvolved in the coupling between ATP hydrolysis and proteolysis.Modulation of Lon activity by cellular metabolites is observed duringamino acid starvation, as the accumulation of cellular polyP results inpolyP binding within the ATPase domain of EcLon, which promotes thedegradation of free ribosomal proteins resulting in the down-regulationof protein synthesis [51,61,67]. In addition, Lon-mediated proteolysis isinfluenced by their interactions with partner proteins. For example,

Fig. 2. The degradation profile of λN and the sequence of the peptide substrate derived from thecleavage sites underlined [71]. B shows the sequence of the fluorogenic peptide used for monitothe λN protein, the fluorophore Abz and the fluorescence quencher nitrotyrosine. An increase

ScLon and HsLon selectively degrade subunits of electron transportchain complexes COX or the F1Fo ATP synthase, when they areunassembled [41,68,69]. Similarly, EcLon selectively degrades theantitoxin protein CcdA that is not assembled with the toxin proteinCcdB [70]. Taken together, these observations suggest that Lon activityis modulated by a variety of reversible protease-ligand and substrateprotein interactions. Fluctuations in the concentration of specific li-gands such as polyP, and changes in the assembly state of proteinsubstrates such as COX or the F1Fo subunits and CcdA, serve as aregulatory switch which activates the Lon protease and allows the cellsto respond rapidly to metabolic needs especially during environmentalstress.

4. Mechanism of protein degradation

The mechanism of Lon-mediated proteolysis has generally beenstudied with protein substrates that are unfolded, oxidatively modified,or with defined structural domains [70–76]. Like other ATP-dependentproteases, Lon is thought to act processively in mediating ATP-depen-dent protein degradation [45,67,71,73]. In general, enzymatic processiv-ity refers to continued catalysis of a series of chemical transformationswithout enzyme dissociation from substrate, as in the case of DNApolymerase. Currently, it remains unclear the extent to which Lon-mediated degradation proceeds by processive peptide bond hydrolysiswithout substratedissociation. Prokaryotic andeukaryotic Lonproteasesdegrade substrates by generating peptide products consisting of ~5–30amino acids [45,63,70,75]. The minimal length of substrates cleaved byLon is not known, however, hydrophobic tetrapeptides are cleaved byEcLon albeit with catalytic efficiencies several orders of magnitudelower than decapeptides or protein substrates [77]. Peptide bond hy-drolysis is thought to occur in a processive linear manner from theamino- to the carboxyl-termini or visa versa. It is possible that proteinsubstrates are sequestered from the bulk solvent by the Lon complex[78] and “processively” degraded by sequential repetitive rounds ofsubstrate binding, cleavage, release and rebinding to the proteolytic site,resulting in small hydrolyzed peptide products. Results also show thatLon does not cleave substrates at a specific peptide consensus sequence;however, it does show preference for hydrophobic residues such asleucine adjacent to the scissile bond [45,70,72,73,75]. In addition to

Lon cleavage profile of λN. A shows the primary sequence of the λN protein, with the Lonring the ATP-dependent peptidase activity of Lon. This peptide contains residues 89–98 ofin the Abz fluorescence is detected upon peptide cleavage by Lon in the presence of ATP.

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rendering cleavage specificity, peptide sequences within an exposedor unstructured region of a substrate may serve to facilitate substraterecognition and interaction, thereby leading to the initiation of de-gradation [70,79]. The molecular details of how such interactions faci-litate protein degradation are not clear and require further mechanisticevaluation.

Like other ATP-dependent proteases, the general paradigm for Lon-mediated proteolysis has been that ATP-dependent substrate unfoldingis a prerequisite for proteolysis. For example, the degradation of foldedproteins by Lon requires ATP hydrolysis. However, if the same protein isless structured or denatured, Lon-mediated degradation can proceedupon ATP-binding alone in the absence of hydrolysis [70]. Nevertheless,the degradation of unstructured proteins and peptides shows maximalefficiency when both ATP binding and hydrolysis occur. For two-component ATP-dependent proteases such as HslUV, Clp and the 26Sproteasome, ATPhydrolysis facilitates the unfolding and translocation ofprotein substrates into a chambered proteolytic core where they aredegraded [80–82]. Recently, a slow peptide translocation step has beendetected in ClpAP, thus it is possible that a similar peptide translocationstep also occurs during Lon catalysis [80]. In the case of Lon, an unfoldedprotein substrate is translocated to the proteolytic active site where itis sequestered until the selective cleavage of scissile peptide bonds iscomplete.

Although many protein substrates of Lon are unfolded prior todegradation, recent work shows that some endogenous substrates ofmitochondrial HsLon are degraded when they are in the folded state[45]. For example, the mitochondrial processing peptidase α subunit(MPP α) is degraded by HsLon only when it is folded, trypsin-resistantand competent for assembly into an active enzyme. By contrast, whenMPPα is unfolded, it is not degradedbyHsLon, even though it is sensitiveto limited trypsin digestion [45]. The sites at which HsLon initiatescleavagewithin foldedMPPα, are surface exposed hydrophobic residuesthat are surrounded by highly charged environments. It is speculatedthat such sites may function as recognition elements or patches, whichspecify selective degradation by HsLon. It is possible that the initiatingcleavages of MPPα by HsLon destabilize the intrinsic folded structure of

Fig. 3. Kinetic model to account for the ATP-depen

the substrate that leads to unfolding and facilitates processive degrada-tion. The finding that HsLon degrades folded proteins suggests that theprotease may function in regulating or terminating the activity of pro-teins, in addition to disposing of abnormal proteins.

5. Kinetic coordination between the ATPase and the proteolyticactivities in EcLon

In the presence of ATP, Lon cleaves protein substrates at mul-tiple sites producing small peptide products. During this process, Lonhydrolyzes ATP, unfolds and/or translocates protein substrates to theproteolytic active site and catalyzes peptide bond cleavage. Whenanalyzing the kinetic mechanism of Lon-mediated degradation ofprotein substrates, it is difficult to determine the specific functions ofATP binding and ATP hydrolysis during each ATPase cycle because thesubstrate contains multiple and diverse sites that are recognized and/or cleaved by Lon. Some substrates such as SulA also contain addi-tional recognition sites within their sequences that are not cleaved byEcLon [75,79]. As such, protein substrates interact with Lon in acomplex manner [75,79,83], which does not allow for a clear corre-lation between ATP hydrolysis and a single peptide cleavage event. Toobviate this complication, hydrophobic tetrapeptides have been usedas model substrates to characterize the peptidase activity of EcLon.However, these peptide substrates inhibit the ATPase activity of EcLonand are degraded at a significantly slower rate as compared to proteinsubstrates [2], and are thus not optimal for evaluating the functionalrelationship between ATP hydrolysis and peptide bond cleavage.

We have therefore developed and utilized a fluorogenic peptidesubstrate (FRETN 89–98, Fig. 2) containing residues 89–98 of the λ Nprotein to determine the timing of ATP hydrolysis and peptide bondcleavage that will allow us to define a minimal kinetic mechanism ofEcLon [77]. Like the full-length λN protein, degradation of FRETN 89–98 EcLon exhibits maximal efficiency when ATP is bound andhydrolyzed. However, degradation of FRETN 89–98 also occurs inthe presence of non-hydrolyzable AMPPNP, albeit with substantiallyreduced efficiency. The kcat/Km value for ATP-dependent cleavage of

dent cleavage of FRETN 89–98 by E. coli Lon.

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FRETN 89–98 by EcLon is 3×104 M−1s−1, which is comparable to thevaluemeasured for the degradation of the native λN protein [77]. As inthe full-length λN protein, FRETN 89–98 is cleaved between cysteineat position 93 and serine at position 94, indicating that this peptidecontains the information needed to confer the same cleavage speci-ficity. Although FRETN 89–98 contains only 11 residues, its ability tostimulate the ATPase activity of Lon is comparable to larger proteinsubstrates. Collectively, these data demonstrate that FRETN 89–98provides a unique tool for studying the kinetic mechanism associatedwith the activation of peptide bond cleavage by ATP binding andhydrolysis by Lon. The mechanistic characterization of FRETN 89–98cleavage by EcLon is a first step in unraveling themechanism bywhichLon catalyzes the degradation of large protein substrates containingmultiple cleavage sites.

To construct a minimal kinetic model for the ATP-dependent pep-tidase reaction mechanism, transient kinetic techniques have beenutilized to detect enzyme bound reaction intermediates generatedalong the ATPase and peptide hydrolysis pathway. The first turnover ofATP hydrolysis and FRETN 89–98 cleavage is monitored by the rapidacid quench and the fluorescent stopped flow techniques, respectively[84]. The EcLon holoenzyme complex exhibits nucleotide binding siteswith different affinities for ATP (Kdb1 μM and 10 μM, respectively,[63,85]). The lower affinity sites (Kd=10 μM) catalyze ATP hydrolysisprior to peptide bond cleavage, and hydrolyze nucleotide with a rateconstant of ~10 s−1, which is 1000-fold faster than the high-affinitysites (Kdb1 μM, kATPase ~0.01 s−1). While occupancy of ATP at the high-affinity ATPase sites support multiple rounds of peptide cleavage, ATPbinding and hydrolysis at both the high- and low-affinity sites arenecessary for optimal cleavage [85].

ADP is bound to Lon with a higher affinity than ATP; however, itsrate constant of release from Lon is higher than the kcat of ATP hy-drolysis in the presence of the FRETN 89–98 peptide or protein sub-strates [85,86]. Furthermore, ADP but not inorganic phosphate (Pi) hasbeen shown to remain bound to Lon after sizing column chromato-graphy [2,63,87]. In addition ADP is a significantly better productinhibitor of Lon as compared to Pi. The Ki of ADP is 0.3 μM against ATP,where no changes in enzyme activity are observed up to 30 mM Pi.Collectively, these results suggest that Pi release occurs prior to ADPrelease; however, the rate-limiting step in the ATPase turnover reac-tion has yet to be determined.

Fig. 4. Structures of peptide boronate

To determine the minimum number of steps involved in the ATP-dependent peptidase reaction as shown in Fig. 3, we have used thefluorescently labeled nucleotides Mant-ATP or Mant-AMPPNP asprobes, which generate changes in fluorescence intensity upon bin-ding to Lon in the presence of peptide substrate. In the absence ofpeptide or protein substrates, Lon catalyzes the hydrolysis of Mant-ATP with comparable Km and kcat values as ATP. Mant-ATP can sub-stitute ATP in activating the peptidase activity of EcLon, and thehydrolysis of Mant-ATP by EcLon is stimulated by the presence ofFERTN 89–98. A dansylated peptide, whose degradation by EcLon isstimulated by ATP, has also been used to monitor peptide bindinginteractions to Lon in the presence of this nucleotide. Upon binding toLon, the fluorescence intensity of the dansylated peptide is enhancedby the exciting the intrinsic Trp residues in the Lon:ATP complex.Using these probes, a two-step mechanism for nucleotide and peptidebinding has been demonstrated [85,88]. In Fig. 3, free EcLon is repre-sented by enzyme form I. For simplicity, the enzyme is shown as adimer with the ATPase domain in green, the SSD domain in blue andthe proteolytic domain in pink. In steps 1 and 2, ATP and peptide bindto Lon independently of one another to yield the ternary complexrepresented by enzyme form II, which undergoes a conformationalchange (step 3) to yield enzyme form III. The timing of ATP andpeptide hydrolysis is deduced by transient kinetic experiments. Thefirst turnover of ATP hydrolysis displays burst kinetics with a rateconstant of 10 s−1 while the hydrolysis of FRETN 89–98 under identicalconditions displays lag kinetics with a rate constant of ~1 s−1 [84].According to the burst amplitude of the ATPase reaction, only 50% ofthe nucleotide bound to the enzyme is hydrolyzed during this timeregime; and the Kd of these ATPase sites is ~10 μM, which correspondsto the low-affinity sites in EcLon. Based on these findings, it is con-cluded that ATP hydrolysis by Lon precedes peptide bond cleavage(step 4, enzyme form IV). The formation of enzyme form IV is furtherimplied by the fluorescence resonance energy transfer (FRET) signalgenerated from the interaction of the dansylated analog of FRETN89–98with the proteasemutant of EcLon S679W,where the proteolyticsite serine at position 679 was replaced with a tryptophan [88]. Theproteolytically inactive mutant displays wild-type ATPase activity and afirst order rate constant of ~10 s−1 was detected for peptide interactingwith S679W. This rate constant agrees with the burst rate constantdetected for the first turnover of ATP hydrolysis at the low-affinity sites

inhibitors against Lon proteases.

733I. Lee, C.K. Suzuki / Biochimica et Biophysica Acta 1784 (2008) 727–735

(Kd=10 μM) [84]. Therefore, the formation of enzyme form IV is likelyfacilitated by the coupled hydrolysis of ATP. Step 5, which generatesenzyme form V, is detected by monitoring the FRET signal between thedansylated peptide and S679W Lon mutant [88]. As the rate constantfor the formation of enzyme form V approximates the pre-steady-statelag rate constant for FRETN 89–98 cleavage by wild-type Lon [84], it isproposed that step 5 contributes to the rate-limiting step in the firstturnover of Lon-mediated peptide bond cleavage in the presence ofATP. Through steady-state product inhibition analyses, Lon has beenshown to undergo an “isomerization step” (step 6) along its catalyticpathway to yield an enzyme form “F” [86]. Hydrolysis of the FRETN89–98 peptide yields two hydrolyzed peptide products. The hydro-lyzed peptide product (PPI) that contains residues 94 to 97 of λNprotein (PPI) is a non-competitive inhibitor against the FRETN 89–98substrate. It is proposed that PPI binds to an enzyme form of Lon (F)that does not bind the FRETN 89–98 substrate. This result could beexplained by a mechanism by which Lon isomerizes following pep-tide hydrolysis, and the post-catalytic enzyme form does not bindFRETN 89–98. This kind of mechanism is generally known as an “iso”mechanism [89]. The existence of an isomerized form of Lon (F) isfurther confirmed by a plateau or upturn in the double reciprocalplots of PPI inhibition versus ATP in the presence of the FRETN 89–98substrate [86]. At high nucleotide and high PPI concentrations,enzyme turnover is inhibited. This inhibition cannot be overcome byincreasing the peptide concentration. Due to this observation, it isconcluded that there is a post-catalytic isomerized form of Lon,designated as F in Fig. 3, which specifically binds the hydrolyzedpeptide product PPI but not the substrate; F is stabilized by thepresence of ATP. Step 7 constitutes the dissociation of hydrolyzedproducts. At this point, it is not clear how the enzyme form F differsfrom the pre-catalytic enzyme form 1. It is conceivable that theenzyme form F proposed in Fig. 3 is a reflection of an enzyme formresponsible for the processive protease activity of Lon. This spe-culation will require additional mechanistic evaluation.

The minimal kinetic scheme illustrated in Fig. 3 can be used as amodel to probe for additional steps in Lon-mediated substrate unfol-ding and cleavage. The detection of a slow step (step 5) prior to FRETN89–98 cleavage is consistent with a “substrate translocation” step alsoobserved with other ATP-dependent proteases [80–82,90,91]. In thedegradation of the full-length λN proteinwhich contains multiple Loncleavage sites, one wonders the same catalytic cycle depicted in Fig. 3is repeated used to catalyze the cleavage of each peptide bond in λN.That is, the hydrolysis of each peptide bond is flanked by a trans-location step. Alternatively, the whole λN protein maybe translocatedand sequestered within the proteolytic site before the peptide bondcleavage initiates. In the latter model, translocation occurs oncewhereas peptide bond cleavage events occurs multiple times via asimilar mechanism proposed for the Clp proteases [80,92].

6. Inhibitors of Lon proteases

As a serine protease, Lon is inhibited non-specifically by serineprotease inhibitors such as phenylmethyl sulfonyl fluoride (PMSF).Generally, the IC50 of Lon inhibitors is within millimolar range [2].Recently, a collection of proteasome inhibitors has been screened foreffects on Lon-mediated proteolysis using the ATP-dependent FRETN89–98 peptidase assay as a detection method [93]. Of all the inhibitorsexamined, the peptide boronate MG262 (Fig. 4), exhibited the highestpotency against Salmonella Lon (Ki =6.6 nM). Derivatization of thecarboxyl terminal of a hydrolyzed product of FRETN 89–98 by Lonwitha boronate moiety also yields an inhibitor exhibiting a Ki of 17 nM(Dansyl 89–98 Abu boronate, Fig. 4). Interestingly, the inhibition byboth peptide boronates towards Lon requires the presence of ATP, andthe boronate adduct targets the proteolytic Ser679 [94]. Presumably,the binding and hydrolysis of ATP is needed to allosterically activatethe proteolytic site of Lon (enzyme form IV in Fig. 3). In addition to

peptide based inhibitors, a recent study using the ATP-dependentdegradation of FITC-labeled casein as a screening assay reveals thatcertain coumarinic derivatives exhibit preferential inhibition towardshuman Lon compared to the 20S proteasome [95]. Based on the struc-tures of these compounds and the general mode of coumarin inhibitionagainst serine proteases, it is proposed that the coumarin moiety reactswith the proteolytic Ser679 as well as a nearby nucleophile. Unlike thepeptide boronates however, the inhibition of Lon by these compoundsdoes not require the presence of ATP. Since these coumarin derivativeshave a low molecular weight, and can be readily modified by chemicalsynthesis, these lead compounds should provide a starting point forstructure activity relation (SAR) studies, whichmay lead to the develop-ment of specific cell-permeable Lon inhibitors.

As bacterial Lon has been shown to be important for the infec-tivity and/or pathogenicity of certain species such as B. abortus andS. typhimurium, compounds that specifically block bacterial but nothuman Lon, hold promise for a new class of anti-microbial agents[27,29–31,93,96]. In addition, the mitochondrial Lon has been impli-cated in the response to oxidative stress and damage to proteins andmtDNA [7,43,97], which has direct relevance to aging. The identificationof specific inhibitors to mitochondrial Lon will provide valuable che-mical genetic tools not only for cataloging the endogenous substrates ofHsLon, but also for defining the physiological functions of this proteasein human health and disease.

Acknowledgements

The work described in the authors' lab was supported by theNational Institutes of Health (GM61095), the Basil O'Connor ScholarsAward - March of Dimes and the American Heart Association to C. K.Suzuki; the National Institutes of Health (GM067172) to I. Lee.

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