However, the active site residues of the protease and ATPase domain are highly conserved in all Lon proteases.. Comparison of the ATPase activity of Lon wild-type from Thermoplasma or Es
Trang 1P R I O R I T Y P A P E R
active site
Henrike Besche and Peter Zwickl
Department of Structural Biology, Max-Planck-Institute of Biochemistry, Martinsried, Germany
A gene with significant similarity to bacterial Lon proteases
was identified during the sequencing of the genome of the
thermoacidophilic archaeon Thermoplasma acidophilum
Protein sequence comparison revealed that Thermoplasma
Lon protease (TaLon) is more similar to the LonB proteases
restricted to Gram-positive bacteria than to the widely
dis-tributed bacterial LonA However, the active site residues of
the protease and ATPase domain are highly conserved in all
Lon proteases Using site-directed mutagenesis we show here
that TaLon and EcLon, and probably all other Lon
pro-teases, contain a Ser-Lys dyad active site The TaLon active
site mutants were fully assembled and, similar to TaLon
wild-type, displayed an apparent molar mass
upon gelfiltration This would be consistent with a hexameric complex and indeed electron micrographs of TaLon revealed ring-shaped particles, although of unknown sym-metry Comparison of the ATPase activity of Lon wild-type from Thermoplasma or Escherichia coli with respective pro-tease active site mutants revealed differences in Km and
Vvalues This suggests that in the course of protein degra-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 3.4.21.53) was the first
ATP-dependent proteolytic enzyme to be identified [1,2] Later
protease La was found to be the product of the
Escherichia coli lon gene
Lon peptidase or protease This can be seen by the
respective entry and listed references in the MEROPS
peptidase database: peptidase family S16 (lon protease
family) (http://merops.sanger.ac.uk/famcards/summary/
s16.htm) [5]
The Lon protease domain is ubiquitously distributed
and mostly fused to different ATPase domains [6]
However, in the genomes of some Bacteria and Archaea,
standalone Lon protease domains are present, but nothing
is known about their biological function In contrast,
plenty of knowledge has accrued about bacterial and
mitochondrial Lon proteases [7–10], where the Lon domain
is linked to an N-terminal AAA+domain which, in turn, is
extended N-terminally by a Lon N-terminal (LAN)
domain [6] Certain bacteria contain a second Lon protease, called LonB, which lacks the LAN domain [6] and is assumed to be soluble [11] Most Archaea contain a LonB homologue, i.e lacking the LAN domain, which contains two transmembrane-spanning regions and was shown to be membrane-bound [12,13]
It has been known for a long time that Lon proteases have an active site serine residue [14], but despite extensive mutagenesis the residual catalytic residues remained elusive [15] Ultimately, mutagenesis studies of a viral noncanon-ical Lon protease lacking the ATPase domain revealed the catalytic Ser-Lys dyad (S-K dyad) for Lon proteases [16]
We set out to mutate the conserved serine and lysine residues in Thermoplasma acidophilum Lon protease (TaLon) to generalize the S-K dyad for ATP-dependent membrane-bound Lon proteases In the course of this work an independent report confirmed the S-K dyad for the E coli Lon protease (EcLon), but without studying mutual regulation of the ATPase and protease domain [17] Based on mutagenesis studies it was proposed that the ATPase domain of EcLon regulates the protease in a unidirectional manner, i.e the mutational inactivated protease domain did not influence the ATPase activity [18] We investigated this aspect in more detail for TaLon and EcLon by detailed analysis of protease active site mutants
Materials and methods
Sequence alignments The active-site regions of Lon protease protein sequences were aligned with [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.de
Abbreviations: AAA + , ATPase associated with various cellular
activities; DDM, dodecyl-b- D -maltopyranoside; EcFtsH, Escherichia
coli FtsH; EcLon, Escherichia coli Lon; LAN, Lon N-terminal; Lonwt,
Lon wild-type; S-K, Ser-Lys; TaLon, Thermoplasma acidophilum Lon;
TaLonwt, Thermoplasma acidophilum Lon wild-type.
Enzyme: endopeptidase La (EC 3.4.21.53).
(Received 19 August 2004, revised 1 October 2004,
accepted 6 October 2004)
Trang 2Generation of active site mutants
Site-directed mutagenesis was performed using the
Quick-Change Kit (Stratagene, La Jolla, CA, USA)
pET22b(+)-TaLon-His6 (S525A and K568A) and Lon
wild-type (pLonwt) (K722A) served as PCR templates The
respective primers are listed with the mutated codons
underlined: S525A, 5¢-CGAGGGAGTTGAAGGAGAC
GCGGCCAGCGTATCAATAGCC-3¢ (sense), 5¢-GGCT
ATTGATACGCTGGCCGCGTCTCCTTCAACTCCCT
CG-3¢ (antisense); K568A, 5¢-CCGGTTGGCGGCGTAAC
CGCAGCGGTTGAGGCAGCTATAGAAGC-3¢ (sense),
5¢-GCTTCTATAGCTGCCTCAACCGCTGCGGTTAC
GCCGCCAACCGG-3¢ (antisense); K722A, 5¢-GCCGA
TGGTGGTTTGAAAGAAGCCCTCCTGGCAGCGCA
TCGCG -3¢ (sense), 5¢-CGCGATGCGCTGCCAGGAG
GGCTTCTTTCAAACCACCGATCGGC-3¢ (antisense)
DNA sequencing (MWG, Ebersberg, Germany)
full-length gene checked all generated plasmids
Expression, purification and enzymic characterization
Wild-type and mutant TaLon proteases were produced, and
the hydrolysis of fluorigenic peptides, FITC-labelled casein
and ATP were assayed as described [13] R Glockshuber
(ETH Zu¨rich, Switzerland) kindly provided the plasmids
pLonwt and pLonS679A [20] Cells of E coli BL21(DE3)
(Novagen, Madison, WI, USA)
-S679A or -K722A) were grown at 37C in 6 L Luria–
Bertani medium containing 100 lgÆmL)1ampicillin At an
attenuance
6 of 0.6 (600 nm) isopropyl thio-b-D-galactoside
was added to a final concentration of 1 mM After further
incubation for 4 h the cells were harvested by centrifugation
(4000 g, 10 min 4C), washed with 50 mM Tris (pH 7.5)
and stored until purification at)80 C Purification of the
EcLon variants was performed as described [20]
Results
Domain organization of Lon proteases
During the sequencing of the T acidophilum genome an
open reading frame (ORF 1081) was identified, which
showed significant sequence similarity to Lon proteases [21]
(Fig 1A,B) TaLon encompasses an N-terminal ATPase
associated with various cellular activities (AAA+domain)
and a C-terminal protease domain, but lacks the N-terminal
a-helical domain inherent in most bacterial and eukaryotic
Lon homologues (Fig 1C) However, the AAA+domain
of TaLon contains an insert of approximately 90 amino acid
residues between the Walker A and Walker B ATPase
signatures [22], which is not present in any bacterial or
eukaryotic Lon sequence The 90-residue insert is found in
all archaeal Lon homologues and is predicted to contain
two consecutive transmembrane helices, suggesting that
archaeal Lon proteases are membrane associated [21]
Gram-positive bacteria, such as Bacilli and Clostridia,
contain a second Lon protease, called LonB which, like the
archaeal Lon homologues, does not contain an N-terminal
a-helical domain (Fig 1C) In contrast with archaeal Lon
proteases the bacterial LonB also lacks the 90-residue insert
harbouring the predicted transmembrane region and is
therefore probably a soluble protease, although this has not been addressed experimentally [11]
Notably, the archaeon Methanosarcina mazei has two lon genes, an archaeal-type lon gene containing two transmem-brane helices and a bacterial-type lon gene including an N-terminal domain (Fig 1) The M mazei archaeal-type longene was not included in a phylogenetic analysis [23] although both genes are also present in the closely related species Methanosarcina acetivorans and M barkeri Simi-larly, the M mazei genome contains both the complete group I and group II chaperonin systems, i.e the bacterial GroEL/GroES and the archaeal thermosome/prefoldin [24] In general, approximately 31% of the ORFs in the genomes of M mazei and its close relatives share the highest similarity with bacterial genes, which is most probably a result of horizontal gene transfer [23] In addition to the bacterial and archaeal lon genes the genome of M mazei contains a noncanonical lon gene (ORF Mm1931), enco-ding for a Lon protease lacking the ATPase domain Noncanonical Lon proteases are also found in bacteria and viruses (Fig 1B,C) A separate study failed to detect the ATP-binding region in the archaeal Lon proteases in sequence comparisons with bacterial homologues and classified the Pyrococcus Lon homologues as ATP-inde-pendent proteases [25] This is easily explained by the fact
Fig 1 Sequence alignment of selected regions of distinct Lon proteases (A) Alignment of the Walker A and Walker B motifs of the AAA +
ATPase-domain Identical residues are marked with #, conserved residues with + (B) Alignment of the S-K dyad of the protease do-main Labelling as described in (A) Essential residues of TaLon pro-tease activity are indicated below (C) Schematic representation of the domain organization of the proteins aligned in (A) and (B) Bs, Bacillus subtilis; Ec, Escherichia coli; IBDVP2, infectious bursal disease virus strain P2; Mm, Methanosarcina mazei; Ta, Thermoplasma acidophilum;
Tk, Thermococcus kodakarensis The UniProt accession numbers of the aligned proteins are: Bs-LonA, P37945; Bs-LonB, P42425; Bs-YlbL, O34470; Ec-Lon, P08177; IBDVP2-VP4, Q82628; Mm-1913, Q8PVP9; Mm-bLon, Q8PSG1; Mm-Lon, Q8Q0K8; Ta-Lon, Q9HJ89; Tk-Lon, Q8NKS6.
Trang 3that archaeal Lon proteases contain a 90-residue insert
between the Walker A and Walker B motifs as mentioned
above, which impairs alignment of the bacterial with the
archaeal ATPase domain After removing the 90-residue
insert the bacterial and archaeal Walker A and Walker B
motifs can be aligned (Fig 1A) Sequence alignment of the
proteolytic core domain shows that the active site S-K dyad
is conserved in archaeal Lon, in bacterial LonA and LonB,
and in noncanonical Lon proteases (Fig 1B) In summary,
the Lon protease domain is an evolutionarily ancient
domain, which is ubiquitously distributed and can be fused
to various other domains [6]
TaLon forms ring-shaped complexes
Overexpression in E coli leads to membrane insertion of
the TaLon protease The isolated membranes were
solu-bilized with detergent, and recombinant TaLon was
purified by Ni2+-nitrilotriacetic acid affinity
chromatogra-phy [13] Subsequent size exclusion chromatograchromatogra-phy
revealed an apparent molar mass of 510 kDa The
transmembrane domain of TaLon is surrounded by a
dodecyl-b-D-maltopyranoside (DDM) micelle with a mass
of 70 kDa Subtraction of the DDM micelle suggests
that six TaLon monomers (72 kDa) assemble into a
hexameric complex (Fig 2A) Consistently, electron
micro-scopic analysis revealed ring-shaped particles of yet
unknown symmetry (Fig 2B) The isolated proteolytic
domain of EcLon was crystallised as a hexameric complex
[26] In contrast, a structural analysis of the yeast
mitochondrial Lon protease by analytical
ultracentrifuga-tion and electron microscopy revealed heptameric
ring-shaped complexes [27] The stoichiometry of the TaLon
complex remains to be determined
S-K dyad active site
Lon from E coli has been known for a long time to be a
serine protease [14,20], but only lately was an active site
lysine residue
7 identified [16,17] These two catalytic residues
are conserved among all archaeal and bacterial Lon
homologues and form an S-K dyad Very recently, the
crystal structure of the hexameric E coli Lon protease
domain has been solved and revealed a unique fold not
observed in other S-K dyad peptidases [26] Only the
catalytic core containing the active site residues is
structur-ally conserved between distinct S-K dyad peptidases [26]
To establish that the Thermoplasma membrane-bound Lon
protease has the same active site, the two conserved residues
were individually mutated to alanine (S525A and K568A)
As with TaLonwt, the S-K mutants were purified as
hexamers (data not shown) sustaining substantial ATPase
activity (Table 1), but no peptidase activity could be
detected (Fig 3A) Consequently, TaLonS525A and
TaLonK568A showed neither dependent nor
ATP-independent proteolytic activity (Fig 3B)
ATPase activity of protease active site mutants
Kinetic analysis of TaLon and its protease active site
mutants revealed that the specific ATPase activity of
Fig 2 Molar mass and electron microscopy of TaLon (A) TaLon (30 mg) was separated on Superdex 200 HiLoad 26/60 column (25 m M Mes pH 6.2, 300 m M NaCl, 5 m M MgCl 2 , 0.5 m M DDM) and detected by UV 280 Molar mass of marker proteins and their respective elution volumes are indicated Gel filtration revealed an apparent molar mass of 510 kDa corresponding to a hexameric complex after subtraction of 70 kDa for the DDM micelle (B) Electron micrograph
of negatively stained (2% uranylacetate) TaLon from the Superdex
200 peak fraction, recorded with a Philips CM 200 FEG transmission electron microscope at 160 kV.
Table 1 ATPase activity of TaLon and EcLon.
Protease K m (m M ) V (P i Æmin)1Ælg)1) TaLonwt 0.196 ± 0.004 0.631 ± 0.003 TaLonS525A 0.176 ± 0.014 0.561 ± 0.012 TaLonK568A 0.111 ± 0.003 0.477 ± 0.003 EcLonwt 0.201 ± 0.004 0.554 ± 0.012 EcLonS679A 0.140 ± 0.014 0.273 ± 0.009 EcLonK722A 0.189 ± 0.011 0.782 ± 0.015
Trang 4TaLonK568A was 25% lower and the affinity for ATP
was increased by 43% when compared with wild-type
TaLon (Table 1) The Km of TaLonS525A remained
unaffected and the specific ATPase activity was only
slightly reduced (10%; Table 1) Thus the mutation of the
catalytic protease residues, especially of Lys568 seems to
enhance ATP binding and concomitantly slow down ATP
hydrolysis In order to generalize this observation the
corresponding E coli lysine mutant was generated
(EcLon-K722A) and purified along with the active site serine
mutant (EcLonS679A) Both mutants were proteolytically
inactive (data not shown) In accordance with our model
that the protease domain might have a regulative influence
on the protease domain, both mutants were affected in their ATPase activity in comparison with the wild-type enzyme The EcLon Ser679 mutant showed only half the wild-type ATPase activity but a 30% higher affinity for ATP, while the lysine mutant was stimulated with respect
to ATP hydrolysis (40%) and hardly affected in its affinity for ATP (Table 1)
Discussion
The Lon protease is ubiquitously distributed and was found
to exist as a standalone protein as well as fused to a AAA+
ATPase-domain (Fig 1C) [6] Whereas the bacterial Lon protease and its homologues in eukaryal organelles are soluble, the archaeal counterpart is membrane-attached [13] Bacterial LonB proteases, like archaeal Lon, lack the LAN domain present in bacterial LonA proteases, but like bacterial LonA, also miss the membrane anchor found in archaeal Lon proteases Taken together the Lon protease domain is highly versatile and can function in different contexts, i.e standalone or fused to an ATPase, and soluble
or membrane-bound
Solubilized TaLon has a molar mass of 430 kDa corresponding to a hexameric complex The same oligome-rization state was revealed recently in the crystal structure of the hexameric EcLon protease domain [26], whereas electron microscopy of yeast mitochondrial Lon proteaese showed a heptameric complex [27]
Using site-directed mutagenesis we established the S-K dyad for the membrane-bound TaLon and confirmed it for the soluble EcLon Subsequently, we characterized the TaLon and EcLon protease-deficient mutants lacking the catalytic serine or lysine residue Kinetic analysis and comparison of their respective ATPase activity revealed a potential regulation of ATPase hydrolysis depending on the proteolytic cycle Though the unidirectional regulation
of the peptidase activity by the ATPase has been described for the EcLon protease several years ago [18], the reciprocal regulation of the ATPase by the protease remained unrecognized, although two reports described impaired ATP hydrolysis of FtsH mutants lacking protease activity Karata et al [28] reported that mutation of the zinc-binding residue His421 in Escherichia coli FtsH (EcFtsH) completely abolished protease activity and reduced the ATPase activity to 23% of wild-type FtsH
In this case, it can be claimed that the prevention of zinc ion binding might lead to structural perturbations that reduce the ATPase activity However, in two independent studies mutations of the catalytic aspartate in the conserved HEAGH motif of EcFtsH [29] and Bacillus subtilis FtsH [30] were generated The conserved aspartate residue activates a water molecule for the nucleophilic attack but does not affect zinc ion binding Again these mutant FtsH proteins were reported to lack proteolytic activity and showed a 20% reduced ATPase activity Taken together with our observation that mutation of the TaLon and EcLon protease active site residues decreases the ATPase activity, we propose that bidirectional crosstalk between the ATPase and peptidase domains is necessary for controlled protein degradation
Fig 3 Proteolytic activity of TaLonwt and the active site mutants
TaLonS525A and TaLonK568A (A) TaLon (46 n M ) and
Thermo-plasma acidophilum proline iminopeptidase (1.83 l M ) was incubated
with 100 l M succinyl-LLVY-7-amido-4-methylcoumarin in assay
buffer (50 m M Mes pH 6.2, 20 m M MgCl 2 , 0.5 m M DDM) at 60 C.
TaLonwt and the ATPase deficient mutant TaLonK63A (compare
[13]) served as control; the wild-type activity was set equal to one (B)
TaLon (116 n M ) was incubated with 5 l M fluorescein
isothiocyanate-casein in the assay buffer with or without 2 m M ATP at 60 C For the
S-K dyad mutants no difference was observed in the presence or
absence of ATP.
Trang 5We thank Ulf Klein (MPI Martinsried) for assistance, Oana Mihalache
(MPI Martinsried) for electron microscopy, Erik Roth (MPI
Martins-ried) for calibration of the Superdex 200 column and Rudi Glockshuber
(ETH Zu¨rich) for providing EcLon expression plasmids We are
indebted to Wolfgang Baumeister (MPI Martinsried) for generous and
continuous support Finally, we want to thank the referees for their
valuable suggestions This work was supported by a grant from the
DFG to Peter Zwickl (Zw58/3-2).
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