In the present study, we demonstrate that isolated recombinant TorD exhibits a magnesium-dependent GTP hydrolytic activ-ity, despite the absence of classical nucleotide-binding motifs in
Trang 1translocation proofreading chaperone induced by domain swapping
David Guymer1, Julien Maillard2, Mark F Agacan1, Charles A Brearley3and Frank Sargent1
1 College of Life Sciences, University of Dundee, Dundee, UK
2 ENAC-ISTE ⁄ Laboratoire de Biotechnologie Environnementale (LBE), EPF Lausanne, Switzerland
3 School of Biological Sciences, University of East Anglia, Norwich, UK
Keywords
Escherichia coli; molecular chaperones;
noncanonical GTPase; TorD protein;
twin-arginine transport pathway
Correspondence
F Sargent, Division of Molecular
Microbiology, College of Life Sciences,
University of Dundee, Dundee DD1
5EH, UK
Fax: +44 1382 388 216
Tel: +44 1382 386 463
E-mail: f.sargent@dundee.ac.uk
(Received 28 July 2009, revised 12
November 2009, accepted 18 November
2009)
doi:10.1111/j.1742-4658.2009.07507.x
The bacterial twin-arginine translocation (Tat) system is a protein targeting pathway dedicated to the transport of folded proteins across the cytoplas-mic membrane Proteins transported on the Tat pathway are synthesised as precursors with N-terminal signal peptides containing a conserved amino acid motif In Escherichia coli, many Tat substrates contain prosthetic groups and undergo cytoplasmic assembly processes prior to the transloca-tion event A pre-export ‘Tat proofreading’ process, mediated by signal peptide-binding chaperones, is considered to prevent premature export of some Tat-targeted proteins until all other assembly processes are complete TorD is a paradigm Tat proofreading chaperone and co-ordinates the mat-uration and export of the periplasmic respiratory enzyme trimethylamine N-oxide reductase (TorA) Although it is well established that TorD binds directly to the TorA signal peptide, the mechanism of regulation or control
of binding is not understood Previous structural analyses of TorD homo-logues showed that these proteins can exist as monomeric and domain-swapped dimeric forms In the present study, we demonstrate that isolated recombinant TorD exhibits a magnesium-dependent GTP hydrolytic activ-ity, despite the absence of classical nucleotide-binding motifs in the protein TorD GTPase activity is shown to be present only in the domain-swapped homodimeric form of the protein, thus defining a biochemical role for the oligomerisation Site-directed mutagenesis identified one TorD side-chain (D68) that was important in substrate selectivity A D68W variant TorD protein was found to exhibit an ATPase activity not observed for native TorD, and an in vivo assay established that this variant was defective in the Tat proofreading process
Structured digital abstract
l MINT-7302371 , MINT-7302377 : TorD (uniprotkb: P36662 ) and TorD (uniprotkb: P36662 ) bind ( MI:0407 ) by molecular sieving ( MI:0071 )
l MINT-7302402 : TorD (uniprotkb: P36662 ) and TorD (uniprotkb: P36662 ) bind ( MI:0407 ) by comigration in non denaturing gel electrophoresis ( MI:0404 )
l MINT-7302387 : TorD (uniprotkb: P36662 ) and TorD (uniprotkb: P36662 ) bind ( MI:0407 ) by cosedimentation in solution ( MI:0028 )
Abbreviations
GAP, GTPase activating protein; IMAC, immobilised metal affinity chromatography; MGD, molybdopterin guanine dinucleotide;
Tat, twin-arginine translocation; TMAO, trimethylamine N-oxide; TorA, trimethylamine N-oxide reductase.
Trang 2The twin-arginine translocation (Tat) system is a
pro-tein-targeting pathway present in the cytoplasmic
membrane of many prokaryotes [1] Tat-targeted
pro-teins are synthesised as precursors with cleavable
N-terminal signal peptides incorporating the distinctive
SRRxFLK ‘twin-arginine’ amino acid motif [2] A key
feature of Tat translocation is the requirement for
physiological substrates to be fully folded before
suc-cessful translocation can occur [3] Escherichia coli
pro-duces 27 Tat substrates [4], the majority of which bind
complex prosthetic groups, fold, activate, and often
oligomerise, in the cytoplasm prior to membrane
trans-location [1,3,5] It is considered that the Tat
translo-case itself may be able to accept or reject pre-proteins
on the basis of their folded state in a ‘Tat quality
con-trol’ process [3] In addition, some proteins are
sub-jected to a chaperone-mediated ‘Tat proofreading’
process in the cytoplasm prior to export Tat
proof-reading involves the direct binding of particular Tat
signal peptides by dedicated chaperones aiming to
pre-vent premature targeting of immature proteins [6,7]
The Escherichia coli trimethylamine N-oxide (TMAO)
reductase (TorA) is a Tat-dependent periplasmic redox
enzyme encoded by the torCAD operon [8] TorA is the
archetypal model Tat substrate synthesised with a 39
residue signal peptide and contains molybdopterin
guanine dinucleotide (MGD) as a single prosthetic
group [9] Acquisition of the MGD cofactor by TorA in
the cell cytoplasm is an essential pre-requisite to
translo-cation of this protein [10] The torD gene product is
involved in cofactor loading into TorA [11–18], and
additionally operates as a Tat proofreading chaperone,
binding directly to the TorA twin-arginine signal peptide
[5,19,20]
TorD belongs to a family of peptide-binding
proteins specifically dedicated to molybdoprotein
assembly [6,7,16,21] Phylogenetic analysis of the TorD
family allows separation of the members into three
broad ‘clades’: the TorD clade, the DmsD clade and
the NarJ clade [16,21] A number of 3D structures
now exist for members of the TorD family and these
all share a unique all a-helical architecture [22–25]
The helices are arranged into two domains (N-terminal
and C-terminal), which are connected by a hinge
region [23] Crystal structures of monomeric forms
have been described [22,24,25]; however, higher-order
oligomers of TorD-like proteins have been observed
[26,27] and the crystal structure of a homodimer has
been solved [23] Dimerisation of the Shewanella
massi-lia TorD protein is driven by ‘domain-swapping’ in
which the N-terminal domain of one protomer packs
onto the C-terminal domain of a second protomer (and vice versa); however, the physiological role of this domain-swapping was not clear [23]
The isolated, recombinant E coli TorD monomer has been shown to bind to the TorA signal peptide
in vitro with an apparent dissociation constant (Kd) of
59 nm [19] Such relatively tight binding led to some speculation as to how binding and release cycles could
be regulated The crystal structure of the Sh massilia TorD homodimer was observed to contain tightly-bound oxidised (and therefore cyclic) dithiothreitol [23] As a result, Hatzixanthis et al [20] hypothesised that this observation could suggest that a common cyc-lic regulatory molecule, perhaps a nucleotide, could normally be bound by TorD Indeed, the monomeric form of the E coli TorD protein was subsequently shown to bind guanine nucleotides with low affinity (apparent Kd 370 lm for GTP) [20], and recent inde-pendent computational analysis predicted a potential GTP-binding site on the DmsD protein from Salmo-nella entericaserovar Typhimurium [24]
In the present study, the relationship between E coli TorD and GTP was investigated We demonstrate that recombinant TorD possesses an intrinsic magnesium-dependent GTP hydrolysis activity in vitro It is revealed that this activity is a property only of the dimeric form of the protein, suggesting that domain-swapping is required to generate the active site In addition, site-directed mutagenesis identifies one resi-due, D-68, that is involved in the substrate selectivity
of this protein
Results
TorD has magnesium-dependent GTPase activity Early ligand-binding experiments [20] and recent bioin-formatic analysis [24] suggested that TorD-like proteins may bind GTP In addition, overproduction of TorD family proteins has been reported to result in the isola-tion of a range of stable homo-oligomeric forms [26,27] In the initial development of our purification strategies, we established that an ion exchange chro-matographic step, immediately following metal affinity chromatography, resulted in isolation of stable mono-meric TorDhis [20,28] This monomeric form was ideal for biophysical studies [19,20] but, despite its ability to bind guanine nucleotides, no nucleotide hydrolysis activity could be detected [20] We therefore decided to explore the possibility that different folding forms of TorDhismay harbour different biological activities
Trang 3First, E coli TorDhiswas overproduced and isolated
by nickel-affinity chromatography Eluate from the
metal affinity column was then assayed directly for GTP
and ATP hydrolytic activity using a malachite green
method for the quantification of free inorganic
phos-phate (Pi) This assay measures free Pi in solution by
spectrophotometric determination of the complex
formed between malachite green, molybdate and free Pi
The initial assay chosen already included magnesium
chloride in the reaction mixture because magnesium ions
are essential for the GTPase activity of the majority of
characterised GTPases [29,30] The assay demonstrated
that TorD exhibits hydrolytic activity towards GTP
(Fig 1A) No Pirelease was detected in the negative
con-trols, which included a sample of the elution buffer used
in the chromatographic experiment and a sample of a
maltose-binding protein isolated in the identical buffers,
and on the same immobilised metal affinity
chromatog-raphy (IMAC) column, as the TorDhis described here
(Fig 1A) In addition, our recombinant TorDhis shows
no hydrolytic activity towards ATP (Fig 1A)
The binding of a magnesium cofactor has long been
established as essential for the activity of canonical
GTP-binding proteins [29,31] The precise role of
Mg2+may vary between enzymes but is clearly
essen-tial for GTP hydrolysis and is almost always required
to allow GTP binding [29,30] To determine the
requirement for magnesium of the GTPase activity of
TorD, the reaction was performed in varying amounts
of MgCl2 (Fig 1B) The GTPase activity of TorD
without added MgCl2 is negligible, establishing that
magnesium is required for TorD GTPase activity
(Fig 1B) Titration of increasing amounts of MgCl2
gradually enhances GTPase activity, with a peak at
approximately 1 mm (Fig 1B) The malachite green
assay was also performed with 1.2 mm MgCl2 in the
presence of 10 mm EDTA In this experiment, the
presence of EDTA completely inhibited the reaction
(data not shown) Furthermore, no other divalent
cations, including manganese, could replace
magne-sium in this assay (not shown)
Finally, the product of the GTP hydrolysis reaction
catalysed by TorD was shown to be GDP by HPLC
analysis (Fig S1) Taken altogether, these data
demon-strate the initial identification of a strictly
magnesium-dependent GTPase activity associated with the IMAC
pool of recombinant TorD protein
GTP hydrolytic activity is a feature of the TorD
homodimer
Having established that GTPase activity was
associ-ated with TorD collected immediately following metal
affinity chromatography, the next step was to further purify the hydrolytic activity by alternative chromato-graphic techniques Size exclusion chromatography using a Superdex 75 column identified a range of molecular mass species present in the nickel-affinity purified TorD sample (Fig 2A) The major peak corre-sponded to an approximate molecular mass of 25.5 kDa, in agreement with the predicted molecular mass of TorDhis of 24.2 kDa, and so likely represents monomeric TorD Lesser protein peaks representing TorD species with approximate molecular mass of
Fig 1 TorD has magnesium-dependent GTPase activity (A) The malachite green assay for in vitro Pi release from nucleotides Reaction mixtures [50 lL aliquots containing 5 m M GTP or ATP, 1.2 m M MgCl 2 and 10 m M Tris–HCl (pH 7.5)] were incubated at
22 C for 24 h containing 0.1 m M of metal affinity chromatography-purified TorD (‘TorD + GTP’ and ‘TorD + ATP’) As controls, an equal amount of a His-tagged maltose binding protein-TorA signal peptide fusion (‘MalE:TorA-SP + GTP’), or protein-free column buffer (‘no protein’), were also assayed in the presence of 5 m M
GTP (B) The GTP hydrolysis reaction is magnesium dependent Reactions containing 0.1 m M of metal affinity chromatography-puri-fied TorD with 5 m M GTP buffered in 10 m M Tris–HCl (pH 7.5) were incubated at 22 C for 24 h in the presence of increasing amounts of MgCl2 Released Pi was assayed by the malachite green method In both (A) and (B), the total phosphate released in each reaction is shown and error bars represent the SEM (n = 3).
Trang 449.0 kDa, similar to the predicted 48.5 kDa of a dimer
of TorDhis, and higher-order oligomers at approxi-mately 85.7 kDa (beyond the linear range of the Superdex 75 column of 3–70 kDa) were also observed (Fig 2A) The presence of TorD oligomers is supported by gel electrophoresis (Fig 2B, C) Denatur-ing SDS–PAGE showed the presence of TorD polypep-tide in all fractions tested, whereas PAGE performed in the absence of SDS allowed a ready visualisation of the different oligomeric forms of TorD present in the higher molecular mass fractions (Fig 2B, C) Magnesium-dependent GTPase activity was restricted to the higher molecular mass fractions, and was completely absent from the monomer form (data not shown)
Cibacron Blue F3G-A is a dye molecule that can
be immobilised to a Sepharose matrix (Blue Sepha-rose HP), and which is able to bind specifically to some nucleotide-binding proteins as a result of its structural similarity to nucleotide cofactors Specifi-cally-bound proteins are then normally eluted by the application of either an amount of cofactor or by increasing the ionic strength Initial experiments with TorD under ‘standard’ conditions for analysing classi-cal nucleotide-binding proteins [32] suggested that the protein did not bind tightly to Cibacron Blue under conditions of low ionic strength, and that nothing was therefore eluted upon application of a high ionic strength solution (data not shown) Surprisingly, how-ever, upon washing the Blue Sepharose column in water after each experiment, a small peak of protein was observed in the eluate To explore this further, the buffer conditions were changed and the metal affinity pool of TorD was applied to a Blue Sepharose column equilibrated in buffer containing 0.5 m NaCl The elu-tion profile revealed that the majority of the sample did not bind to the Cibacron Blue dye (Fig 3A) However, upon switching from the relatively high salt
A
B
C
Fig 2 Oligomeric forms of E coli TorD can be readily identified (A) Elution profile of a pool of TorD derived from metal affinity chro-matography applied to a Superdex 75 (10 ⁄ 30) size exclusion col-umn at 0.5 mLÆmin)1in 50 m M Tris–HCl (pH 7.5) and 200 m M NaCl Values for the apparent molecular mass of peak proteins were cal-culated using control proteins of known molecular mass (not shown) (B) Equivalent volumes (5 lL) of the protein fractions indi-cated were diluted 1 : 1 in either Laemmli or native sample buffer and subjected directly (unboiled) to SDS–PAGE (top panel) and non-SDS–PAGE (bottom panel) on 12.5% (w ⁄ v) polyacrylamide gels Protein bands were visualised with Coomassie Brilliant Blue R-250 stain (C) Equivalent amounts (3.6 lg) of protein in each indicated fraction were separated directly (unboiled) by SDS–PAGE (top panel) and non-SDS–PAGE (bottom panel) on 12.5% (w ⁄ v) poly-acrylamide gels and stained with Coomassie R-250.
Trang 5concentration buffer (0.5 m) to a very low ionic
strength solution (pure water in this case), a small
peak of protein was eluted from the column (Fig 3A)
SDS–PAGE and western analysis revealed that this
fraction contained TorD protein (Fig 3B, C), and
TorD isolated in this way is referred to in the present
study as ‘TorDBlue’
The TorDBlue protein peak isolated by the ‘reverse’
Blue Sepharose chromatography protocol was analysed
for GTPase activity using the malachite green assay (Fig 3D) Very interestingly, TorDBlue demonstrated GTPase activity, whereas the ‘flow-through’ fraction of TorD showed negligible activity (Fig 3D) Control experiments using a column fraction containing no protein also showed no activity (Fig 3D) Very unusu-ally, therefore, all of the GTPase activity was bound
to the Cibacron Blue column in the presence of 0.5 m salt (something that a ‘canonical’ nucleotide-binding protein would not do) and, subsequently, all of the GTPase activity could be eluted in a solution of very low ionic strength (again not the typical behaviour of
a classical nucleotide-binding protein)
The relative purity of the TorDBlue preparation was analysed further by SDS–PAGE and MS Both the TorDhis fraction (from the initial metal affinity chro-matography pool) and the TorDBlue preparation were separated by SDS–PAGE and the proteins were visual-ised using the most sensitive silver staining method available (Fig 3E) This method revealed a single
A
B
C
D
E
1400
1200
1000
800
600
Ve (ml)
400
200
0
10 20 30 40 50 60
27
Fig 3 TorD GTPase activity can be isolated by Cibacron Blue affin-ity chromatography (A) Unusual behaviour of TorD on Cibacron Blue affinity media A sample of 0.5 m M metal affinity chromatogra-phy-purified TorD was applied to a 1 mL HiTrap Blue column, attached to an FPLC system, in 0.5 M NaCl-containing buffer at
1 mLÆmin)1 Bound proteins were eluted in a single step to pure water (B) Protein fractions from the unbound flow-through (‘FT’) or single 1 mL fractions eluted in water (numbered 35–38) were diluted 1 : 1 in either Laemmli or ‘native’ sample buffer and sub-jected (unboiled) to SDS–PAGE (top panel) and non-SDS–PAGE (bottom panel) Three microlitres of the flow-through sample was used to give an equivalent amount of protein loaded compared to that of 36 mL fraction ( 1.1 lg) Gels were stained with Coomas-sie R-250 (C) A western immunoblot was carried out on 6 ng sam-ples of the original metal affinity-purified material (‘IMAC’), the unbound flow-through (‘FT’), and pooled fractions 36–38 Proteins were mixed with Laemmli disaggregation buffer and heat-treated at
100 C for 2 min before being separated by SDS–PAGE, blotted onto nitrocellulose, and challenged with an anti-TorD serum (1 : 10 000 dilution) (D) Analysis of the ‘TorD Blue ’ fractions for GTPase activity Protein samples (1.2 lg) of fractions eluted in water from the Cibacron Blue Sepharose column were subjected to the malachite green GTPase assay The fraction at 33 mL contained
no detectable protein and was assayed as an internal control to establish that incubation with the column buffers alone did not facil-itate Pirelease Error bars indicate the SEM (n = 3) (E) Pooled pro-tein samples (4 lg) from the original metal affinity column (‘IMAC’) and the combined eluate from the Cibacron Blue affinity column (‘Blue’) were mixed with Laemmli disaggregation buffer and heat-treated at 100 C for 2 min before being separated by SDS–PAGE using Bio-Rad (Bio-Rad, Hercules, CA, USA) pre-cast 15% (w ⁄ v) polyacrylamide gels Proteins were visualised using the Silver-quest(Invitrogen) silver-staining kit and, where indicated, the reac-tion was stopped after 2 or 4 min.
Trang 6strong band in each fraction corresponding to the
TorD protein (Fig 3E) In addition, the TorDBlue
preparation was subjected to MS analysis (Fig S2)
Recombinant TorD was found to the dominant species
in this preparation and was intact, except for partial
modifications to the initiator methionine (Met-1),
which are common in bacterial systems (Fig S2)
Thus, from a total of 45 mg of recombinant TorDhis
isolated by metal affinity chromatography and applied
to the Cibacron Blue column, 2.3 mg of the TorDBlue
protein harbouring all of the GTPase activity was
recovered
Further analysis of the two different TorD pools by
SDS–PAGE and native PAGE suggested that the key
difference lay in the oligomeric state of the proteins
The TorD population that failed to bind to the
Ciba-cronTM Blue migrated at a low apparent molecular
mass under native conditions (Fig 3B), displaying
sim-ilar behaviour to the monomer form of TorD
charac-terised by molecular exclusion chromatography
(Fig 2) However, the GTPase-active TorDBlue clearly
comprised oligomeric TorD (Fig 3B) The precise
olig-omeric state of the TorDBlue species was therefore
investigated by analytical ultracentrifugation
Sedimentation velocity analysis was performed on a
sample taken from the initial nickel-affinity
chroma-tography and this material was shown to contain
predominantly monomeric TorD with detectable
quan-tities of dimer and higher oligomeric species (Fig 4A)
By contrast, however, analytical ultracentrifugation
reveals that the GTPase-active TorDBlue species
iso-lated here contains no monomeric TorD at all
(Fig 4B) Instead, it was unequivocally established
that TorDBlue is dominated by the homodimeric form
of the protein (Fig 4B) Trace amounts of
higher-order oligomeric species are also present in this sample
(Fig 4B)
Taken together, these data strongly indicate that
gross overproduction of recombinant TorD results in a
mixed population comprising numerous different
oligo-meric forms A small sub-population of proteins
(approximately 5% by mass of the total) was found to
adopt a stable homodimeric conformation that
har-bours a magnesium-dependent GTPase activity
Kinetics of the GTPase reaction
The Cibacron Blue affinity chromatography approach
presented the opportunity to study the kinetics of GTP
hydrolysis catalysed by the active TorD fraction For
these experiments, Pi release from GTP hydrolysis by
TorDBlue was assayed continuously using the
Enz-Chek Phosphate Assay Kit (Invitrogen, Carlsbad,
CA, USA) This coupled assay is based on the proto-col described by Webb and Hunter [33] whereby, in the presence of Pi, 2-amino-6-mercapto-7-methylpurine riboside is converted by purine nucleoside phosphory-lase to ribose-1-phosphate and 2-amino-6-mercapto-7-methylpurine The product is detected by an increase
in A360 The GTPase activity of TorDBluein a range of GTP concentrations was assayed in 96-well plates, the
Pi-dependent product measured continuously at A360 and a curve plotted of the initial velocities (V0) against the substrate concentration (Fig 5A) The best fit to
Fig 4 The homodimer form of TorD dominates the GTPase-active fraction Sedimentation velocity analytical ultracentrifugation profiles
of (A) the TorD pool immediately following metal affinity chroma-tography at 0.5 mgÆmL)1, rmsd = 0.024682, f ⁄ f 0 = 1.199770, and (B) the TorD Blue pool immediately following Cibacron Blue aff-inity chromatography at 0.5 mgÆmL)1, rmsd = 0.024537, f ⁄ f 0 = 1.201629 Samples at 0.25 and 0.75 mgÆmL)1were also analysed (not shown) and gave similar profiles.
Trang 7these data resulted in a sigmoidal curve (Fig 5A),
indicative of a co-operative substrate binding and
hydrolysis model with non Michaelis–Menten kinetics
To extract the kinetic parameters from these data, a
Hill plot (Fig 5B) was drawn of V0)(Vmax⁄ V0) against
log10[GTP], using an estimate for Vmax of 39 lmÆmin)1
obtained from the linear plot (Fig 5A) A line of best
fit revealed the values for Km and the Hill coefficient,
h A value of 2.13 for h was obtained, which indicates
positive co-operativity of GTP binding and hydrolysis
by the TorD homodimer TorD was calculated to
hydrolyse GTP with a Km of 1.42 mm and a Kcat of 3.9 min)1, both contributing to a very low specificity constant (Kcat⁄ Km) of 45.77 M)1Æs)1
TorD residue D-68 confers substrate specificity Analysis of the primary amino acid sequence data suggests that TorD differs substantially from any canonical GTPase enzymes For example, regulatory GTP hydrolases (‘G-proteins’) all possess a conserved guanine nucleotide-binding domain, with the five polypeptide loops that comprise the guanine nucleo-tide-binding site being the most highly conserved elements that define the GTPase superfamily [31] These five loops are designated G-1 to G-5, with G-1, G-3 and G-4 being present in all canonical GTPases [34,35] G-1 corresponds to the ‘Walker A motif’ [36] and includes the consensus sequence Gx4GK[S⁄ T] G-2 (DxnT) is a structurally mobile element and, in many GTPases, GTP-binding alters the conformation of this loop, bringing a conserved threonine essential for
Mg2+ co-ordination into a position to facilitate GTP hydrolysis [35] G-3 corresponds to the ‘Walker B motif’ [36] and includes the consensus sequence Dx2G [35] Although the G-1 and G-3 consensus motifs are found in many nucleotide-binding proteins, it is the G-4 motif that provides the specificity for guanine nu-cleotides The characteristic sequence motif of G-4 is [N⁄ T][K ⁄ Q]xD [34,37,38] and it is often preceded by a stretch of four hydrophobic or nonpolar amino acids [35] Finally, G-5 ([T⁄ G][C ⁄ S]A) is less well conserved than the other motifs and cannot always be unambigu-ously identified from primary sequence data alone [35] TorD lacks each of the canonical G-1 and G-3 motifs that are considered essential for GTP recogni-tion and hydrolysis However, examinarecogni-tion of the amino acid sequence reveals a potential candidate for
a G-4 guanine specificity motif in TorD Praefcke et al [39] described the human guanylate-binding protein that has guanine specificity conferred by a G-4 motif with the sequence TLRD In addition, the homologous GBP in chicken contained a TVRD motif at this posi-tion This is of particular interest because the E coli TorD possesses a TVRD tetrapeptide at positions 65–
68, which is predicted to be located on an exposed loop between helix 4 and helix 5
The residues of this putative G-4 motif were sub-jected to site-directed mutagenesis The focus was resi-due D-68, the final resiresi-due in the TVRD motif, because examples have been described where substitu-tions at this position have altered the substrate speci-ficity of canonical GTPases [34,37,39–42] In classical GTPases, the aspartate is considered to form a
hydro-A
B
Fig 5 Kinetics of GTP hydrolysis by the TorD homodimer (A) A linear
plot of initial reaction rate, V0(released Pi; l M Æmin)1) against GTP
con-centration (m M ) plotted using ORIGIN software (OriginLab Corporation,
Northampton, MA, USA) Aliquots (10 l M ) of a pooled fraction of
TorD Blue isolated by Cibacron Blue affinity chromotagraphy were
asayed in a 96-well plate format at 37 C with shaking in the presence
of increasing amounts of substrate and the formed product measured
continuously at A360using the EnzCheck assay (B) A Hill plot of the
same data shown in (A) converted into log (V 0 – V max ⁄ V 0 ) against
log[GTP] Vmaxwas estimated as 39 l M Æmin)1 The straight line was
calculated by Microsoft Excel (Microsoft Corp., Redmond, WA, USA)
using liner regression: y = 2.1302x – 0.3237 (R 2 = 0.9478) From
these data, the Hill coefficient (equal to the gradient of the line),
h = 2.13, Km [10 (y ⁄ )h)] = 1.42 mM, K
cat = 3.9 min)1, and Kcat⁄ K m = 45.77 M )1Æs)1.
Trang 8gen bond with the amino group at position 2 of the
guanine base, which is absent from the xanthine base
[39,40] Thus, D-68 was substituted by asparagine and
the hydrolytic activity of the TorDD68Nvariant towards
GTP and XTP was measured using the malachite green
assay (Fig 6A, B) TorDD68N was unaltered for
GTPase activity, although this variant demonstrated
enhanced XTPase activity (Fig 6A, B) To test for an
increased affinity for xanthine nucleotides by a different
means, the standard malachite green assay was
modi-fied to perform an assay measuring the effect of
compe-tition of a ten-fold excess of XMP on the GTPase
activities of the native protein and the TorDD68N
vari-ant The GTPase activity of TorDD68Nwas found to be
inhibited to a greater degree than the native TorD by
the presence of excess XMP (Fig 6B)
Examples exist of naturally-occurring GTPases
where the G-4 consensus aspartate is substituted by
tryptophan, and these are able to hydrolyse ITP in
addition to GTP [34,37,41] A TorD D68W variant
was therefore tested for its ITPase and ATPase activity
(Fig 6C) The TorDD68W protein showed no obvious
increase in ITPase activity compared to the native
pro-tein, which could clearly hydrolyse ITP already
(Fig 6C) More interestingly, however, TorDD68W was
observed to possess a hydrolytic activity towards ATP
(Fig 6C), a substrate that native TorD was unable to
recognise (Figs 1A and 6C) Taken together, these data
implicate the TorD ‘G-4’ motif in playing a key role in
substrate selectivity for this enzyme, especially with
respect to the ability of the enzyme to distinguish
between GTP and ATP
A TorD D68W variant is defective in the Tat
proofreading process
The physiological role of TorD residue D-68 was
tested in vivo TorD has two physiological functions
that can be independently measured and assays have
been developed to study the overall biosynthesis of the
TorA enzyme, as well as the isolated Tat proofreading
activity First, the ability of the torD gene to rescue
TMAO reductase activity in a DtorD mutant when
expressed in trans was explored A chromosomal
dele-tion strain (FTD100) was transformed with
pUNI-PROM derivatives encoding native TorD and
TorDD68W The strains were grown anaerobically in
LB supplemented with glycerol and TMAO and the
benzyl viologen-linked TMAO reductase activity of
periplasmic fractions was assayed (Fig 7A) TorDD68W
was observed to support assembly of the periplasmic
TMAO reductase activity to a level equivalent to
native TorD (Fig 7A)
120%
A
B
C
100%
80%
60%
40%
20%
0%
120%
100%
80%
60%
40%
20%
0%
4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
wtTorD + GTP
wtTorD + GTP wtTorD + A
TP wtTorD + ITP D68W + GTP D68W + A
TP D68W + ITP
wtTorD + GTP + XMP
Elapsed time (h)
wtTorD – GTP wtTorD – XTP D68N – GTP D68N – XTP
Fig 6 TorD residue D68 controls substrate specificity (A) The GTPase and XTPase activities of 0.1 m M ( 0.122 mg) native TorD and the D68N variant purified by immobilised metal affinity chroma-tography were assayed by the malachite green method in 50 lL reac-tions containing either 5 m M GTP or XTP, 1.2 m M MgCl2,and 10 m M
Tris–HCl (pH 7.5) Reactions were incubated at 22 C for 24 h The results are shown as a percentage of the native TorD GTPase activ-ity (B) A timecourse competition ⁄ inhibition assay of GTPase activity using 0.1 m M metal affinity chromatography-purified TorD and TorD D68N in 100 lL reactions containing 10 m M Tris–HCl (pH 7.5), 1.2 m M MgCl2and 5 m M GTP ± 50 m M XMP Phosphate release was quantified at 2 h intervals by withdrawing 10 lL aliquots and subjecting those to the malachite green assay (C) Nucleotide hydro-lysis activities of 0.1 m M samples of metal affinity chromatography-purified TorD and TorDD68Wassayed by the malachite green method Each 50 lL reaction contained 5 m M of either GTP, ATP, or ITP as well as 1.2 m M MgCl2and 10 m M Tris–HCl (pH 7.5) Reactions were incubated at 22 C for 24 h and the results are shown as a percent-age of GTPase activity exhibited by native TorD In all cases, the error bars represent the SEM (n = 3).
Trang 9Next, a specific assay for Tat proofreading was
employed Jack et al [5] developed an assay based on
a strain (RJ607) producing a
TorA-signal-peptide-HybO fusion protein Cells producing the TorA-TorA-signal-peptide-HybO
fusion have impaired hydrogenase-2 activity because
assembly of the enzyme is disrupted; however,
co-expression of active torD restores the Tat proofreading
of this enzyme and so rescues hydrogenase-2 activity
in the mutant strain RJ607 was transformed with a pUNIPROM vector encoding TorD and TorDD68W, grown anaerobically in LB supplemented with glycerol and fumarate, and benzyl viologen-linked hydroge-nase-2 activity was assayed in whole cells (Fig 7B) Interestingly, TorDD68W was observed to have a reduced Tat proofreading activity in vivo
Discussion
The crystal structure of Sh massilia TorD is a homod-imer formed through 3D domain swapping [23] It has been established in the present study, in common with other TorD homologues [26,27], that E coli TorD can also be purified in a range of stable oligomeric forms, suggesting that oligomerisation may be a characteristic feature of the TorD family Most significantly, this work has now established the biochemical relevance of the dimerisation exhibited by TorD The E coli TorD homodimer displays an intrinsic specific GTPase activ-ity, whereas the monomer form remains completely inactive In terms of kinetics, the sigmoidal curve of
V0versus [GTP] is indicative of a co-operative binding and hydrolysis model, and the Hill coefficient (h) of 2.13 implies a strongly positive co-operative binding model whereby binding and hydrolysis of GTP at one site enhances the affinity (or activity) towards GTP at other sites The Hill coefficient also enables an estima-tion of the number of hydrolytic sites present in the enzyme because current dogma states that h cannot be greater than the number of ligand-binding sites present within the molecule This suggests either that the active form of TorD could contain at least three active sites,
or that the active oligomeric form could be larger than dimeric Indeed, the TorD protein from Sh massilia has been previously observed as stable trimers [27] Note that h provides only a minimum estimate for the number of binding sites Haemoglobin, for example, shows a Hill coefficient for oxygen binding varying in the range 1.7–3.0, but clearly possesses four binding sites [43,44]
The discovery of an enzymatic activity associated with the domain swapped TorD dimmer, which is absent from the monomer, brings E coli TorD sharply into line with other biological systems that utilise domain swapping to regulate activity For example, glyoxylase I of Pseudumonas putida is a metastable domain-swapped dimer with two Zn2+ cofactors that also exists as a monomer with a significantly reduced activity and only a single Zn2+cofactor [45] An addi-tional example is the bleomycin resistance protein,
Fig 7 Residue D68 is involved in the Tat proofreading process.
Physiological activity of the TorDD68W variant (A) E coli strain
FTD100 (DtorD) was transformed with a pUNIPROM vector
expressing either torD (‘TorD’), or the D68W mutant (‘D68W’), and
grown anaerobically in LB containing 0.5% (v ⁄ v) glycerol and 0.4%
(w ⁄ v) TMAO before intact cells were assayed for TMAO:BV
oxido-reductase activity (B) E coli strain RJ607 (/torA::hybO, DhybA)
was transformed with a pUNIPROM vector expressing either torD
(‘TorD’), or the D68W mutant (‘D68W’), and grown anaerobically in
LB containing 0.5% (v ⁄ v) glycerol and 0.4% (w ⁄ v) fumarate before
whole cells were assayed for hydrogen:BV oxidoreductase activity.
In all cases, the error bars represent the SEM (n = 3).
Trang 10which sequesters two molecules of the bleomycin
anti-biotic within two crevices formed at the interface of
the domain swapped dimer [46] Equally intriguing is
the T7 endonuclease I, representing one of the most
striking examples of a domain-swapped dimer, which
comprises a composite catalytic site containing
ele-ments from both polypeptide chains [47,48] It is
tempting to speculate that TorD dimerisation generates
an analogous composite active site, which would
enable the dimer to acquire a completely new
biologi-cal function through domain swapping Indeed, such
acquisition of a novel function would be consistent
with the theory that domain swapping provides a
model for the evolution of oligomeric enzymes [49,50]
Although the mechanism of GTP hydrolysis by the
TorD dimer remains to be elucidated, there are aspects
of this activity that are mirrored in canonical
nucleo-tide hydrolases For example, the GTPase activity of
TorD is dependent upon the presence of magnesium
ions and this is clearly in line with the requirements of
the classical GTPases [29,30] Magnesium often
func-tions as a phosphate ligand in canonical GTPases in
addition to a variety of proposed activities involved in
the actual hydrolysis reaction [30,31,51] Although the
exact nature of the GTP-binding site remains to be
determined, it is clear that TorD represents a novel
family in this regard This is reinforced by the
observa-tion that active E coli TorD behaves very unusually
on Cibacron Blue affinity media, eluting only under
conditions of low ionic strength, perhaps suggesting
hydrophobic interactions between the cofactor and
protein dominate Interestingly, a more typical G-4
‘guanine specificity’ loop was identified in TorD, and
the D-68 side chain unequivocally was demonstrated
to regulate substrate access to the active site(s) The
E coli TorD sequence in question, 65-TVRD-68, is a
close match to the G-4 consensus [N⁄ T][K ⁄ Q]xD
[34,37,38] This motif is conserved in the TorD and
DmsD clades of the wider TorD family and is always
located in an unstructured loop between helices 4 and
5 (Fig 8) The final aspartate in the TVRD
tetrapep-tide is occasionally naturally replaced by glutamine or
glutamate (Fig 8G), although these side chains should
still able to confer guanine specificity in classical
GTP-ases [41] Note also that the TorD G-4 motif is often
immediately followed by another conserved aspartate
(Fig 8G), which could also have a role in determining
nucleotide specificity
This TorDD68Wvariant was unaffected in its GTPase
activity but, in addition, showed a new ATPase activity
The fact that this protein was defective in the Tat
proof-reading process is intriguing Why would ATP
hydroly-sis not be able to substitute for GTP hydrolyhydroly-sis in vivo?
Because the ATP hydrolysis reaction catalysed by the variant TorD form is obviously slower, and the concen-tration of ATP in the cell is higher than GTP, it is possi-ble that ATP is inhibiting the Tat proofreading function
of TorDD68Win vivo.
In an attempt to identify catalytic residues essential for GTP hydrolysis, extensive mutagenesis was con-ducted on E coli torD (Fig S3) based on recent bio-chemical and in silico experiments [5,19,20,24] However, from the 17 different TorD variants tested, none were found to be completely devoid of in vitro GTPase activity (Fig S3) TorD variants Q7L, as orig-inally identified by Buchanan et al [19], and F41W [20] showed the lowest hydrolytic activity towards GTP (Fig S3), and these side-chains are in close prox-imity to the G-4 loop (Fig 8)
What is the purpose of the GTPase activity of TorD? GTP hydrolysis is often used to regulate pro-cesses where multiple proteins must function co-ordi-nately [52] and the maturation of TorA is certainly a complex process involving the co-ordination of the two distinct functions of TorD with the folding of TorA and possible interactions of both proteins with the MGD biosynthetic apparatus [12] The concentration
of GTP in the cytoplasm of an exponentially growing
E coli cell has been estimated at approximately 0.9 mm [53] This is below the Km of GTP hydrolysis
by TorD and perhaps suggests that TorD has only a low level of this activity in vivo Most GTPases bind very stably to GTP and have a very low intrinsic level
of GTPase activity, and also require the action of GTPase activating proteins (GAPs), or the interaction with a specific effecter, to catalyse the hydrolysis of GTP The transition from GTP- to GDP-bound forms can therefore be limited by the intrinsic rate of GTP hydrolysis, by the action of a GAP, or by interaction with a cognate target GTPases governed by these fac-tors are often referred to as ‘clocks’, ‘switches⁄ adap-tors’ and ‘sensors’, respectively [31] The turnover number of TorD for GTP of 3.9 min)1 is within the range expected for classical GTPases [31] but is very low in general terms, and it is possible that TorD may function as a ‘clock’ with the low rate of GTP hydro-lysis coinciding with the maturation rate of TorA Another possibility is that arginines of the signal pep-tide, or perhaps residue R22 of TorD [20], may be contributing an ‘arginine finger’, thus activating the GTPase activity in response to signal binding Arginine fingers have been observed within the Ffh-FtsY com-posite active site and provided by the Ras and Rho GTPase GAPs [54–56] However, the experiments conducted in the present study were unable to demon-strate an increase in the rate of GTP hydrolysis in