Intrinsic GTPase activity of a bacterial twin-arginine
translocation proofreading chaperone induced by domain
swapping
David Guymer
1
, Julien Maillard
2
, Mark F. Agacan
1
, Charles A. Brearley
3
and Frank Sargent
1
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: [email protected]
(Received 28 July 2009, revised 12
November 2009, accepted 18 November
2009)

MI:0407)bymolecular 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.
FEBS Journal 277 (2010) 511–525 ª 2009 The Authors Journal compilation ª 2009 FEBS 511
Introduction
The 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-

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 (K
d
)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 K
d
 370 lm for GTP) [20], and recent inde-
pendent computational analysis predicted a potential
GTP-binding site on the DmsD protein from Salmo-
nella enterica serovar Typhimurium [24].

may harbour different biological activities.
The E. coli TorD homodimer has GTPase activity D. Guymer et al.
512 FEBS Journal 277 (2010) 511–525 ª 2009 The Authors Journal compilation ª 2009 FEBS
First, E. coli TorD
his
was 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 (P
i
). This assay measures free P
i
in solution by
spectrophotometric determination of the complex
formed between malachite green, molybdate and free P
i
.
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 P
i
release 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,

2
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 SuperdexÔ 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 TorD
his
of 24.2 kDa, and so likely represents
monomeric TorD. Lesser protein peaks representing
TorD species with approximate molecular mass of

D. Guymer et al. The E. coli TorD homodimer has GTPase activity
FEBS Journal 277 (2010) 511–525 ª 2009 The Authors Journal compilation ª 2009 FEBS 513
49.0 kDa, similar to the predicted 48.5 kDa of a dimer
of TorD
his
, and higher-order oligomers at approxi-
mately 85.7 kDa (beyond the linear range of the
SuperdexÔ 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).
CibacronÔ Blue F3G-A is a dye molecule that can
be immobilised to a Sepharose matrix (Blue Sepha-
roseÔ 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

acrylamide gels and stained with Coomassie R-250.
The E. coli TorD homodimer has GTPase activity D. Guymer et al.
514 FEBS Journal 277 (2010) 511–525 ª 2009 The Authors Journal compilation ª 2009 FEBS
concentration 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 ‘TorD
Blue
’.
The TorD
Blue
protein peak isolated by the ‘reverse’
Blue Sepharose chromatography protocol was analysed
for GTPase activity using the malachite green assay
(Fig. 3D). Very interestingly, TorD
Blue
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

)
400
200
0
12 17 22 32 37
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 CibacronÔ
Blue affinity media. A sample of 0.5 m
M metal affinity chromatogra-
phy-purified TorD was applied to a 1 mL HiTrapÔ Blue column,
attached to an FPLC system, in 0.5
M NaCl-containing buffer at
1mLÆmin
)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-

TorD protein (Fig. 3E). In addition, the TorD
Blue
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 TorD
his
isolated by metal affinity chromatography and applied
to the Cibacron Blue column, 2.3 mg of the TorD
Blue
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-
cron
TM
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 TorD
Blue
clearly
comprised oligomeric TorD (Fig. 3B). The precise olig-
omeric state of the TorD
Blue

these experiments, P
i
release from GTP hydrolysis by
TorD
Blue
was assayed continuously using the Enz-
Chek
Ò
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 P
i
, 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 A
360
.
The GTPase activity of TorD
Blue
in a range of GTP
concentrations was assayed in 96-well plates, the
P
i
-dependent product measured continuously at A
360
and a curve plotted of the initial velocities (V
0

)(V
max
⁄ V
0
) against
log
10
[GTP], using an estimate for V
max
of 39 lmÆmin
)1
obtained from the linear plot (Fig. 5A). A line of best
fit revealed the values for K
m
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 K
m
of 1.42 mm and a K
cat
of
3.9 min
)1
, both contributing to a very low specificity
constant (K
cat
⁄ K
m

2
G
[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, examination 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 residue 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

log[GTP]. V
max
was estimated as 39 lMÆmin
)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, K
m
[10
(y ⁄ )h)
] = 1.42 mM, K
cat
= 3.9 min
)1
, and K
cat
⁄ K
m
=
45.77
M
)1
Æs
)1
.
D. Guymer et al. The E. coli TorD homodimer has GTPase activity

(Fig. 6C). The TorD
D68W
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, TorD
D68W
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
TorD
D68W
. The strains were grown anaerobically in

2.0
1.5
1.0
0.5
0.0
02
wtTorD + GTP
wtTorD + GTP
wtTorD + ATP
wtTorD + ITP
D68W + GTP
D68W + ATP
D68W + ITP
wtTorD + GTP + XMP
D68N + GTP D68N + GTP + XMP
46
Elapsed time (h)
[P
i
] mM
81012
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 mM MgCl
2,

518 FEBS Journal 277 (2010) 511–525 ª 2009 The Authors Journal compilation ª 2009 FEBS
Next, 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-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 TorD
D68W
,
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, TorD
D68W
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

domain-swapped dimer with two Zn
2+
cofactors that
also exists as a monomer with a significantly reduced
activity and only a single Zn
2+
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 TorD
D68W
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).
D. Guymer et al. The E. coli TorD homodimer has GTPase activity
FEBS Journal 277 (2010) 511–525 ª 2009 The Authors Journal compilation ª 2009 FEBS 519
which 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-

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 TorD
D68W
variant 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 hydrolysis 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 TorD
D68W
in 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-

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
The E. coli TorD homodimer has GTPase activity D. Guymer et al.
520 FEBS Journal 277 (2010) 511–525 ª 2009 The Authors Journal compilation ª 2009 FEBS
response to addition of excess TorA signal peptide
(data not shown). There is currently no evidence for
the requirement of a specific GAP for TorD GTPase
in vitro, although it remains a possibility that such
may exist in vivo and further work to identify potential
interacting partners will be required to investigate this.
The recent finding that TorD can bind the molybde-
num cofactor, and may also interact with MobA in
addition to two sites on apoTorA [12], suggests that

Plasmids
Overproduction of TorD
his
was achieved from plasmid
pQI-TorD [19], which incorporates a C-terminal hexahisti-
dine tag onto the protein. For constitutive expression of
torD, plasmids pSU-TorD [5] and pUP-TorD [5], deriva-
tives of pSUPROM and pUNIPROM [5], respectively, were
used. Point mutations were introduced into the torD
sequence by site directed mutagenesis using the Quikchange
protocol (Stratagene, La Jolla, CA, USA).
Protein production and purification methods
The host strain for TorD
his
overproduction was E. coli C43
(DE3) [57] harbouring pREP4 (lacI
+
, Kan
R
; Roche Diag-
nostics, Basel, Switzerland). Protein production was per-
formed in LB medium supplemented with appropriate
antibiotics at 37 °C. Cells were grown with shaking until D
600
of  0.5 was reached followed by a further 3 h under
‘inducing’ conditions in the presence of 1 mm (final) isopro-
pyl b-d-thiogalactoside. Following overproduction, cells were
harvested by centrifugation, resuspended in 20 mm Tris–HCl
(pH 7.6) and 1 mm dithithreitol, and broken by two passages
through a French pressure cell at 8000 psi. Debris and unbro-

IMAC-purified TorD
his
was applied in this buffer. Follow-
ing re-equilibration, bound protein was eluted in a single
step from loading buffer to deionised water.
Protein concentrations of purified preparations were
determined by measuring A
280
and applying molar extinc-
tion coefficients of 30 940 MÆcm
)1
for TorD
his
and
66 350 MÆcm
)1
for FMalE-TorA
his
.
Protein analysis
The SDS–PAGE method was as described by La
¨
mmli
[60] and non-SDS-PAGE followed the same protocol but
omitted SDS at every stage. Western immunoblotting was
performed as described previously [61].
Analytical ultracentrifugation (sedimentation
velocity)
Proteins were prepared as 1.0 mgÆmL
)1

reaction available commercially as a kit. For the discontin-
uous assay, 0.1 mm samples of purified protein were incu-
bated (typically overnight) in a reaction mixture containing
10 mm Tris–HCl (pH 7.5), 5 mm GTP and 1.2 mm MgCl
2
at 37 °C. On completion, samples were diluted 250-fold in
water and mixed with malachite green solution [0.7 m HCl,
0.3 mm malachite green oxalate, 8.3 mm Na
2
MoO
4
, 0.05%
(v ⁄ v) Triton X-100] in a 1 : 1 ratio. Following a 15 min
incubation at 22 °C, A
630
was measured and compared with
a phosphate standard curve prepared using identical
solutions.
For the continuous assay, the activity of 10 lm TorD
Blue
was determined using the EnzChek
Ò
Phosphate Assay Kit
The E. coli TorD homodimer has GTPase activity D. Guymer et al.
522 FEBS Journal 277 (2010) 511–525 ª 2009 The Authors Journal compilation ª 2009 FEBS
(Invitrogen). Reactions were started by the addition of
protein and phosphate release was measured continuously
using a Synergy 2 Plate reader (BioTek Inc., Winooski, VT,
USA) set to measure A
360

tion of reduced benzyl viologen was then assayed as
described previously [65].
Acknowledgements
We thank Tracy Palmer (Dundee) for useful discus-
sions and Martin Zoltner (Dundee) for help with anal-
ysing the kinetic data. Mass spectrometry was carried
out by Kenneth Beattie and Samantha Kosto of the
Fingerprints Proteomics Facility at the University of
Dundee. This work was funded in the UK by the
BBSRC through a Doctoral Training Grant awarded
to the University of Dundee, and J.M. was supported
by the Swiss National Science Foundation.
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