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Legionella phosphotyrosine phosphatase activity towards MAPKs
1
The Legionella pneumophila effector Ceg4 is a phosphotyrosine phosphatase that attenuates activation of
eukaryotic MAPK pathways
Andrew Quaile1, Peter J Stogios1, Olga Egorova1, Elena Evdokimova1, Dylan Valleau1, Boguslaw
Nocek2, Purnima S Kompella3, Sergio Peisajovich3, Alexander F Yakunin1, Alexander W
Ensminger4, Alexei Savchenko1,5*
1Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON,
Canada.
2Structural Biology Center, Advanced Photon Source, Argonne National Laboratory.
3Department of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada,
4Department of Biochemistry, Department of Molecular Genetics, University of Toronto, Toronto, ON,
Canada
5Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine,
University of Calgary, Calgary, AB, Canada
Running title: Legionella phosphotyrosine phosphatase activity towards MAPKs
.
To whom correspondence should be addressed: Dr. Alexei Savchenko. 5Department of Microbiology,
Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary, 3330 Hospital
Drive NW, Calgary, Alberta, Canada; Telephone: (403)-210-7980; E-mail: alexei.savchenko@ucalgary.ca
Keywords: Legionella pneumophila, crystallography, phosphotyrosine, protein phosphatase, peptide array,
p38 MAPK, bacterial effector.
ABSTRACT
Host colonization by Gram-negative
pathogens often involves delivery of bacterial
proteins called “effectors” into the host cell. The
pneumonia-causing pathogen Legionella
pneumophila delivers more than 330 effectors
into the host cell via its type IVB Dot/Icm
secretion system. The collective functions of
these proteins is the establishment of a replicative
niche from which Legionella can recruit cellular
materials to grow while evading lysosomal fusion
inhibiting its growth. Using a combination of
structural, biochemical, and in vivo approaches,
we show that one of these translocated effector
proteins, Ceg4, is a phosphotyrosine phosphatase
harboring a haloacid dehalogenase–hydrolase
domain. Ceg4 could dephosphorylate a broad
range of phosphotyrosine-containing peptides in
vitro and attenuated activation of MAPK-
controlled pathways in both yeast and human
cells. Our findings indicate that L. pneumophila’s
infectious program includes manipulation of
phosphorylation cascades in key host pathways.
The structural and functional features of the Ceg4
effector unraveled here, provide first insight into
its function as a phosphotyrosine phosphatase,
paving the way to further studies into L.
pneumophila pathogenicity.
Host colonization by Gram-negative
pathogens often involves delivery of specific sets
of bacterial proteins called “effectors” into the
host cell by specialized secretion systems. Acting
in concert, effectors secure the pathogen’s
survival and replication, via manipulation of host
processes and the establishment of subcellular
environments that favor the pathogen. Disruption
of effector translocation (1-3) results in
attenuation of intracellular growth, highlighting
the essential role of effectors in pathogen-host
interactions. Despite significant progress in
identification of effector arsenals in bacterial
genomes and in defining the molecular functions
of individual effectors, many remain
uncharacterized, necessitating further studies into
their role in pathogenesis. The composition and
number of effectors varies dramatically between
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Legionella phosphotyrosine phosphatase activity towards MAPKs
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different pathogens. For example, plague and
intestinal disease-causing Yersinia species
encode less than ten effectors delivered by the
type III secretion system (4) while the similar
secretion system in Shigella spp. is involved in
translocation of close to 30 effectors (5). Notably,
sequence-related effectors are found in pathogens
with very diverse invasion strategies suggesting
that these effector families are involved in
common host manipulation tactics.
A causative agent of severe pneumonia in
humans, the Legionella pneumophila genome
encodes over 330 effector proteins, that are
translocated via the Dot/Icm (defect in organelle
trafficking/ intracellular multiplication), type
IVB secretion system (e.g. (6-11), or see (12) for
a recent review). Legionella’s effectors account
for more than 10% of its proteome (13) and
represents the largest effector set known to the
bacterial world. In their natural habitat of fresh
water reservoirs Legionella spp. invade diverse
amoebae by preventing formation of the
phagolysosome (14) followed by modification of
the newly co-opted compartment into an
organelle ideal for intracellular replication of the
bacteria, the Legionella containing vacuole
(LCV) (15). The ability to apply the same
invasion strategy to invade human alveolar
macrophages raises the intriguing possibility that
Legionella’s effectors target host processes that
are conserved between distant eukaryotic phyla.
The sheer number of Legionella effectors and
their apparent functional redundancy makes their
functional characterization particularly
challenging (16).
Studies into the function of bacterial
effectors suggested that these pathogenic factors
demonstrate unparalleled abilities to manipulate
a wide spectrum of host cell’s processes
including facilitating alterations in cytoskeletal
rearrangement (17,18), vesicular trafficking
(19,20), signal transduction (21,22) and
transcription regulation (23,24). To achieve these
effects, effectors are often involved in post-
translational modification (PTM) of specific host
proteins. A key regulation mechanism in both
eukaryotic and bacterial cells, PTMs typically
involve enzymatic covalent modification of
targeted proteins at specific residues, which
affect that protein’s activity, localization or
interactions thus triggering a change in protein
function. Effector proteins can possess not only
the PTM activities found in the bacterial world
but also ostensibly exclusively eukaryotic ones.
Of particular prevalence, bacterial effectors have
evolved to mimic the activity of ubiquitin protein
ligases, which control the final step in the
eukaryote-specific PTM involving attachment of
the ubiquitin polypeptide to targeted proteins,
usually resulting in this protein’s degradation by
the proteasome (25). Effectors with this PTM
activity have been identified in the arsenals of
many bacterial pathogens, including E. coli
(26,27), Shigella flexneri (24,28), Salmonella
spp. (26,29) and Legionella pneumophila (30,31).
The most common PTM in eukaryotic
cells is phosphorylation, in which a phosphate
group from a donor molecule such as ATP onto
hydroxyl functional groups on residues (serine,
threonine or tyrosine) of the targeted protein
(32,33). This PTM is catalyzed by kinases and the
human genome encodes several hundred protein
kinases divided into tyrosine and serine/threonine
specific enzymes (34). This PTM mechanism is
involved in most if not all known human cell
processes.
One of the best-studied examples of
phosphorylation-controlled signaling is mediated
by Mitogen-Activated Protein Kinases (MAPKs).
MAPKs are highly conserved Ser/Thr protein
kinases that have been extensively studied for
their central roles in mediating signal
transduction of extracellular stimuli to the
appropriate biological response (35). MAPKs are
activated by dual phosphorylation of threonine
and tyrosine residues in a Thr-X-Tyr motif
located in their activation loops by upstream
kinases (MAPK kinases, or MAPKKs) (36). In
turn, MAPKs are responsible for phosphorylating
and activating downstream MAPK activated
protein kinases (MAPKAPKs or MKs). These
downstream targets elicit activation of processes
including regulation of stress response,
proliferation, differentiation and apoptosis. Their
control of processes highly relevant to bacterial
infections as well as their careful regulation and
conservation among eukaryotes have made
MAPKs attractive targets for bacterial effectors;
Icm/Dot substrates LegK1-4 share
homology to eukaryotic protein kinases and
activation of MAPKs in response to Legionella is
well documented (37-39). Furthermore, while
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Legionella phosphotyrosine phosphatase activity towards MAPKs
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phosphorylation of SAP/JNK, ERK1/2 and p38
are seen at early time-points even in translocation
deficient mutants (39), sustained activation of
SAP/JNK and p38 is reliant on effector
translocation (38).
Although protein phosphatases have not
been previously detected amongst Legionella’s
complement of effectors (40) precedent for this
mechanism amongst other effectors of other
species undoubtedly exists; Yersinia pestis YopH
is a phosphotyrosine phosphatase capable of
removing the pTyr signal through hydrolysis,
thereby muting its activity (41), while Shigella
flexneri OspF is a phosphothreonine lyase that
irreversibly dephosphorylates the activating
threonine of MAPKs (42). A recent analysis of
the effector repertoire in the causative agent of Q
fever, Coxiella burnetii also identified effectors
Cbu1676 and Cbu0885 as phosphatases targeting
the MAP kinase pathway in the eukaryotic host
surrogate S. cerevisiae (43). S. cerevisiae
possesses five main MAP kinase pathways
regulating filamentation, cell wall integrity,
sporulation, mating and hyperosmosis, with the
latter two pathways, controlled by Fus3 and Hog1
respectively, being most broadly conserved
among diverse eukaryotic organisms (44). The
closest human homologues of Fus3 and Hog1, are
ERK2 and p38. The first indication of Cbu1676
and Cbu0885 function came from in silico
analysis that pointed to the presence of the
haloacid dehalogenase-like (HAD-like) domain
in these bacterial proteins (43). Proteins
containing HAD-like domains have a broad range
of activities including dehalogenase,
phosphonatase and phosphomutase activity and
although the majority characterized thus far are
phosphatases and ATPases (45,46), protein
phosphatase activity has only been observed in
eukaryotes (47-50).
All HAD-like domains share a common
overall fold featuring a core Rossmann fold,
consisting of at least two pairs of α-helices that
sandwich the core five-stranded parallel β-sheet
in the order ‘54123’, with a squiggle and flap
motif at the end of β1-strand (45,46). The
common molecular architecture between HAD
superfamily members includes four (I to IV)
highly conserved sequence motifs co-localized to
the active site. Motifs I and IV feature conserved
aspartate residues that coordinate the Mg2+ ion
required for catalysis. In addition to squiggle and
flap motifs, HAD-like domains typically contain
an insertion to the catalytic core domain called a
cap domain, which controls active site access and
is involved in substrate binding (51-53). Despite
these recognizable sequence motifs, significant
variation in substrate specificity and activity of
the HAD-like protein family necessitates detailed
structural analysis and rigorous substrate
specificity studies.
In addition to the Coxiella effectors
mentioned above, putative HAD-like domains
have been identified in the uncharacterized
effectors Lpg0096 (also known as Ceg4 / “co-
regulated with the effector encoding genes 4”),
Lpg1101 and Lpg2555 from L. pneumophila
(43). Here, using x-ray crystallography and
biochemical activity screening, we show that
Ceg4 is an atypical HAD-like phosphotyrosine
phosphatase able to attenuate the activation of
MAP kinases in both human and yeast cells.
These results indicate that L. pneumophila
facilitates its infectious program by manipulation
of phosphorylation cascades of key pathways in
its host cells.
RESULTS
Legionella effector Ceg4 demonstrates
phosphotyrosine specific phosphatase activity in
vitro–The Dot/Icm-dependent translocation of L.
pneumophila Ceg4 protein encoded by the
lpg0096 gene was previously demonstrated using
CyaA fusion translocation assays (8). Our
sequence analysis using Phobius (54) suggested
that in addition to the N-terminal haloacid
dehalogenase-like (HAD-like) domain
mentioned above, this effector contains two C-
terminal transmembrane (TM) helices (residues
266-289 and 295-320). As no TM signatures were
detected in Lpg1101 or Lpg2555 we hypothesize
that Lpg0096/Ceg4 may be a member of a
functionally diversified family that relies of
localization to appropriately direct its activity
toward the host. Using data from two recent large
scale comparative genomics studies of Legionella
species (55), we performed phylogenetic analysis
of Ceg4 with the 33 other putative HAD domain-
containing effectors (Figure 1). Ceg4, Lpg1101
and additional 12 homologues formed a distinct
clade corresponding to Legionella orthologue
group LOG_02908, while Lpg2555 and five
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other effectors were localized in a distinct group.
Comparative sequence analysis across the Ceg4
containing clade indicated that sequence
conservation among these effectors was highest
within the HAD-like domain, with significant
sequence variation in their C-terminal domains.
Lani_0822 from L. anisa was the sole exception
to this observation, possessing an additional 50
residues at both the N and C termini. Lpg1101 is
also unique within LOG_02908, having
homology to the phosphatidylinositol 4-
phosphate binding domain of SidM/DrrA-like
(56,57) at its C-terminal. Most notably however,
eight out of the 14 Ceg4 homologues possessed
two TM domains in their C-terminal portion,
suggesting that localization to the host membrane
is an important and common feature to this subset
of HAD-like effectors in Legionella.
To confirm the general activity of the
Ceg4 HAD-like domain, we purified an N-
terminal fragment spanning residues 1 to 193 of
L. pneumophila Ceg4 (see Materials and Methods
for details). In line with predictions, the Ceg4[1-
193] fragment demonstrated robust phosphatase
activity against the generic phosphatase substrate
para-nitrophenylphosphate (pNPP) (58). Highest
reaction rates were observed between pH 6.5 and
8, with activity dropping markedly above pH 8.
Ceg4 also demonstrated a strict requirement for
Mg2+ metal ions (Supplementary data, Figure
S1B,C,D). Expanding on these results, we tested
Ceg4[1-193] for activity against a library of 94
phosphorylated metabolic substrates allowing for
querying a broad range of possible specificities
(58). In these assays, Ceg4[1-193] demonstrated
the highest activity toward phosphotyrosine
(Figure 2C, see also Supplemental Figure S1E).
To determine if Ceg4[1-193] is active against
protein substrates, we screened for its ability to
remove the phosphate group from a selection of
53 phosphopeptides, chosen for their importance
to signaling in Saccharomyces cerevisiae, which
has been successfully used as a model eukaryotic
system in characterization of bacterial effector
functions including those from Legionella
(30,59). This set included pSer-, pThr- and pTyr-
containing sequences. Consistent with our
previous results, Ceg4[1-193] demonstrated
robust phosphatase activity against nine diverse
peptides comprising the full pool of pTyr-
containing sequences in this substrate array
(Figure 1E, Supplemental Figure S1F), and
moreover, at levels 4 to 5 times higher than for
pSer or pThr peptides. Combined, this data
showed that Ceg4 is a phosphotyrosine specific
phosphatase active against peptide substrates.
Crystal structure of HAD-like domain
provides molecular insight into phosphatase
activity of Ceg4–To gain further insight into the
molecular function of Ceg4, we determined the
crystal structure of the Ceg4[1-208] fragment to
1.88 Å by the single wavelength anomalous
dispersion (SAD) method (see Table 1 for x-ray
crystallographic statistics). The final structural
model spanned Ceg4 residues 1-204 and a portion
of the N-terminal fusion tag sequence
(GQENLYFQG) corresponding to the TEV
protease cleavage site (Figure 3A). In addition to
a core Rossmann-like fold, the HAD-like domain
of Ceg4 included a cap subdomain consisting of
three α-helices and two long loops inserted
between the D9-X-D11 “squiggle motif” and the
α1 helix (Figures 2A and 2B). The location of the
cap subdomain insertion in relation to the
conserved motifs I and II of the core Rossman
fold classified it as a C1 cap (46). Such C1 caps
were previously identified in cN-III nucleotidase
(60), Eya2 protein tyrosine phosphatase (61) and
MDP-1 sugar phosphatase (62,63). However,
according to our analysis, the Ceg4 cap
subdomain did not show any significant structural
similarity with these or any other C1 caps of
structurally-characterized HAD-like domains
(Supplementary Figure S2).
In addition to overall structural similarity
to other Rossmann-like folds, the core fold of
Ceg4 possessed several conserved features
consistent with the canonical motifs of other
HAD-like phosphatases, such as D9 and D11
(motif I), T103 (motif II), K135 (motif III), D157
and D158 (motif IV) (Figure 3C). Collectively,
these residues formed a small pocket ~350 Å3 in
volume, with N20 from the cap subdomain
forming a “lid” over the pocket (Figure 3D).
Inspection of the active site appears to confirm
the correct positioning of D9 and D11 for their
putative functions as the nucleophile and general
acid/base residues respectively. The active site
also contained a Mg2+ ion that was coordinated by
D9, D158 and three ordered water molecules; the
position of the magnesium ion is conserved with
those of mono- and divalent ions bound to other
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HAD-like phosphatases (60-62). The active site
also contained a Cl- ion associated with the
sidechain of K135, the backbone of K104 and two
ordered water molecules; its position is conserved
with the position of sulfate or phosphate ions
trapped in the active site of other crystallized
HAD-like phosphatases (60-62). Finally, the
active site also contained a highly-coordinated
water that interacted with the F8, D9, D11, T103
and the Cl- ion.
To confirm the involvement of these
residues with Ceg4 catalytic activity, we
performed site-directed mutagenesis and tested
the resultant Ceg4[1-193] mutants in vitro for
activity towards pNPP and pTyr substrates as
described above. In accordance with their
predicted contributions to catalysis, the D9A,
D11A, D11N, D157A, D158A and D162A
mutations abrogated phosphatase activity,
validating their essentiality for the catalytic
function of this protein (Supplementary Figure
S3). Inspection of the molecular packing in
the Ceg4[1-208] crystal lattice did not reveal
significantly extensive contacts indicative of
oligomerization. However, by size exclusion
chromatography (Supplementary Figure S4A),
we observed a mixture of monomeric and dimeric
species. Consistent with this, we observed that
the nine residues that we resolved of the N-
terminal fusion tag comprising the TEV protease
cleavage sequence (G(-8)Q(-7)E(-6)N(-5)L(-
4)Y(-3)F(-2)Q(-1)G(0)) contacted the active site
of the adjacent molecule in the crystal lattice
(Supplementary Figure S4B). This peptide
adopted an extended, nearly β-strand-like
conformation (Supplementary Figure S4C).
Notably, the tyrosine residue in the tag sequence
deeply bound in the active site pocket, with its
hydroxyl group forming a network of interactions
including hydrogen bonds with the sidechain of
D11, the bound Cl- ion, and with two ordered
water molecules (Supplementary Figure S4C).
Other interactions included hydrogen bonds
between the sidechain of Q(-1) of the tag and
E40, between the backbone amide of F(-2)
residue of the tag and N20 of the C1 cap, between
the sidechain of K104 and the backbone carbonyl
of Q(-6), and R(-7) of the tag formed two
interactions (with E165 and the backbone
carbonyl of G133). The C1 cap also interacts
with the fusion tag peptide via hydrophobic
interactions between Y43 and Y(-3) and F24 and
Q(-1) and a hydrogen bond between E40 and Q(-
1). Given Ceg4 specificity toward pTyr
peptides, we hypothesized that our observed
binding of the tyrosine from the N-terminal
fusion tag may be representative of Ceg4
interactions with its substrate. To further
examine this, we modified the N-terminal fusion
TEV cleavage sequence to match the sequence
one of pTyr carrying peptides – Q(-7)M(-6)T(-
5)G(-4)Y(-3)V(-2)S(-1)T(0) identified as a Ceg4
substrate in our peptide array screening and
representing the activation loop sequence of the
yeast Hog1 MAP kinase (64). Hog1 is involved
in a signaling pathway regulating yeast
hyperosmotic adaptation (64) and is a close
homologue of human p38 MAP kinase, a
pathway previously implicated in Legionella
pathogenesis (38,39,65). As mentioned
previously, Coxiella HAD-like effectors
efficiently modulated the activity of other yeast
MAP kinases prompting us to hypothesize that
our in vitro activity results may also be indicative
of Ceg4 activity against MAPK kinases.
The structure of the tag-modified
Ceg4[1-208]HOG1p fragment was solved to 1.9 Å
by Molecular Replacement (Table 1). This crystal
structure was almost identical to the original
Ceg4[1-208]TEV site structure described above, and
superimposed with RMSD of less than 0.3 Å
across the entire protein backbone. In this crystal
structure, we resolved the positions of the six
Hog1-derived residues T(-5)G(-4)Y(-3)V(-2)S(-
1)T(0) from the modified fusion tag. As with the
Ceg4[1-208]TEV site structure, the crystal packing
of Ceg4[1-208]HOG1p showed the N-terminal
peptide interacted with the active site of the
adjacent Ceg4[1-208]HOG1p molecule (Figure 4A),
with the Y(-3) residue bound deeply in the pocket
(Figure 4B). The HOG1p peptide also adopted
an extended β-strand like conformation and its
structure was strikingly similar with that of the
TEV cleavage site peptide, most especially across
residues -5 through 0 (RMSD 0.5 Å over the six
matching Cα atoms) (Figure 4C). As we could
resolve only a shorter region of this peptide, we
were able to identify fewer interactions between
this peptide and Ceg4[1-208], and those were
similar the ones observed in the Ceg4[1-208]TEV
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site structure (i.e. the interactions between Y(-3)
and the active site, N20 and the backbone amide
of residue (-2) of the tag, and hydrophobic
interactions between Y43 and Y(-3) plus F24 and
residue -1).
Next, we compared the position of the
tyrosine from the expression tags and the active
site configuration of the Ceg4[1-208] active site
with other structurally characterized HAD
phosphatases. This analysis showed that the
fusion tag tyrosine hydroxyl group adopts a
position that is 3.4 Å from the bound chlorine
atom, which itself occupied the same general
position as phosphate or phosphate analogs (i.e.
phosphate bound to E. coli YrbI (PDB 3I6B, (66))
or beryllium trifluoride bound to Eya2 (PDB
3HB0, (61)) (Supplementary Figure S2B). The
position of the Ceg4[1-208]-bound magnesium
ions were also conserved in position with other
HAD-like phosphatases (Supplementary Figure
S2B). This analysis indicates that the active site
configuration observed in the Ceg4[1-208]
crystal structures is a good approximation of the
position of a phosphotyrosine substrate bound to
this enzyme.
Overall, our structural analysis revealed
a compact active site in the HAD-like domain of
Ceg4[1-208] restricted by a unique cap motif.
This active site is able to accommodate the
phosphotyrosine residue from a peptide substrate,
the position of which can be gleaned from the
conformation of the fusion tag in the Ceg4[1-208]
crystal lattice.
Ceg4 shows activity against Hog1 and
Fus3 MAP kinases in Saccharomyces cerevisiae–
According to our phosphopeptide library
screening, Ceg4[1-193] demonstrated equally
robust activity against peptides QMTGpYVSTR
and GMTEpYVATR representing the activation
loops of yeast Hog1 and Fus3 MAP kinases,
respectively (Fig. 1C). To test if in vitro activity
of the Ceg4[1-193] fragment against yeast MAP
kinase phosphopeptides is representative of in
vivo activity of this effector, we tested the ability
of full-length Ceg4 to affect the activation of
Hog1- and Fus3-controlled pathways in a yeast
model system. For this we used two S. cerevisiae
strains engineered to express fluorescent reporter
proteins (Stl2-BFP or Fus1-GFP) upon activation
of Hog1 or Fus3 controlled pathways,
respectively (see Material and Methods for
details). We expressed full-length Ceg4[1-397] or
the Ceg4[1-208] fragment in these S. cerevisiae
strains and measured the overall fluorescence
signal from 10,000 cells. S. cerevisiae strains
overexpressing Ceg4 showed significant
reduction of activation of both Hog1 and Fus3
activated pathways (42% and 56%, respectively),
as compared to the control strain carrying an
empty vector (Figure 5A). The strain expressing
the Ceg4[1-208] fragment showed a reduced
ability to suppress MAPK activation in the case
of Fus3-controlled activation compared to the
strain expressing the full-length effector. In
contrast, the expression of the Ceg4[1-208]
fragment resulted in further decrease in Hog1-
controlled activation compared to the strain
expressing the full-length effector. Based on
these results, Legionella Ceg4 is implicated in
regulation of host MAPK controlled pathways as
demonstrated by reduced expression of
fluorescent reporters for both Fus3 and Hog1. In
addition, our data suggested that Ceg4 activity
against the Fus3 pathway is dependent on the C-
terminal region of the effector that contains the
membrane-spanning elements, pointing to the
potential role of this domain for in vivo specificity
of Ceg4.
Next, to test the link between Ceg4
phosphatase activity and MAPK regulation we
probed the effect of individual Ceg4 active site
residue substitutions on the ability of this effector
to dampen the activation of Hog1-controlled
pathways in yeast. The Ceg4 residues targeted by
this analysis were chosen based on their direct
involvement in phosphatase catalytic activity
(D11, K135, D158, D162 and), and their
participation in forming the active site by the cap
subdomain (K17, S18, N20, V23, F24, E26 and
Y43) and the core HAD-like domain (K104,
E108, E131, T161 and N186) (Figure 5B and C).
In line with our in vitro activity results,
alanine substitution of the conserved D11, K135,
D158 and D162 residues directly involved in
catalytic activity of the HAD-like domain
resulted in abrogation of Ceg4 suppression of the
hyperosmotic stress response. This observation
confirmed our hypothesis that Ceg4-triggered
dampening of MAPK activation is directly linked
to the phosphatase activity of its HAD-like
domain. Substitution of active site pocket
residues V23, F24, Y43 and K104 also had a
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negative impact on the ability of Ceg4 to control
MAPK activation with V23D substitution
resulting in complete loss of activity. In contrast,
substitutions of Ceg4 K17, E26, E108, E131 and
T161 which are located distally from the active
site and N20 which partially covers the entrance
to the catalytic pocket did not significantly affect
its activity against Hog1 activated pathway.
These observations are consistent with previously
observed mechanisms of catalysis of HADs and
furthermore indicate the involvement of certain
cap domain residues in substrate recognition.
Combined, these results clearly linked
Ceg4’s in vivo activity as a regulator of MAPK
pathways with its HAD-like domain phosphatase
active site and identified individual residues
involved in catalysis and substrate interactions.
Ceg4 localizes to HeLa endoplasmic
reticulum and attenuates MAPK p38 activation in
vivo–Having determined that Ceg4 is able to act
on conserved eukaryotic MAP kinases, and
having also established a possible role of C-
terminal region of the Ceg4 effector in this
activity, we were interested in identifying the
subcellular localization of Ceg4 in human cells
and in determining if the MAPK dampening
activity extended to human MAP kinases.
To that end, human HeLa cells were
transfected with constructs expressing wild type
Ceg4, the catalytically inactive variant Ceg4D9A,
Ceg4[1-207] and Ceg4[208-397] fragments each
fused to GFP. In keeping with the presence of the
predicted transmembrane regions in the C-
terminal portion of Ceg4, the full length (both
wild type and D9A mutant) and Ceg4[208-397]
fragments demonstrated specific perinuclear
localization, while the construct Ceg4[1-207]
which lacks the C-terminal region containing the
TM domain showed diffuse localization
throughout the cell (Figure 6A). Given the
differential importance of the C-terminal domain
to dampening S. cerevisiae pheromone and
hyperosmolarity responses we posit that this
portion of Ceg4 is critically important to its
function in the host cell. We therefore sought to
more specifically determine the sub-cellular
localization of Ceg4. HeLa cells transfected with
N-terminally GFP tagged Ceg4D9A,
counterstained with ER-tracker dye demonstrated
that Ceg4 co-localizes predominantly with
endoplasmic reticular structures.
With both structural and biochemical
analyses indicating MAPK activation loop
sequences as potential targets of Ceg4
phosphatase activity, we also tested its ability to
perform this function on the closest human
homologue of Hog1, the p38 MAPK. HEK293t
cells transfected with either Ceg4 or Ceg4D9A
were tested for changes in the phosphorylation
state of human p38 MAPK upon stimulation with
either TPA or anisomycin. Western blot analysis
for both total p38 and phospho-p38 (Figure 6C)
showed that while the total levels of p38 are the
same in cells expressing the wild type and
catalytically-inactive mutant, cells harboring
wildtype Ceg4 demonstrated a significant
reduction of signal corresponding to
phosphorylated p38 compared to the same signal
in cells carrying the Ceg4D9A mutant.
Combined, our structural and functional
analysis has identified Legionella Ceg4 as a
bacterial HAD protein tyrosine phosphatase that
is able to attenuate the MAP kinase responses in
both human and yeast cells in vivo, and does so
via removal of the phosphate moiety from
phospho-tyrosine in their activation loops that are
critical to their activation.
DISCUSSION
Translocation of effector proteins inside
the host cell is an important and common strategy
adopted by many Gram-negative bacteria
including important human pathogens. This
necessitates the functional characterization of
specific effectors as a necessary step in
understanding of host-pathogen interactions and
for the development of novel anti-bacterial
therapies. Here, we show the conserved
Legionella effector Ceg4 can modulate the
phosphorylation state of eukaryotic MAP kinases
through its HAD-like phosphatase domain and
we clarify the molecular structure of this domain,
providing key molecular details into this effector.
Ceg4 is one of three predicted HAD-like
domain containing effectors in the Legionella
pneumophila genome that are known to be
translocated by the Dot/Icm system. According to
our analysis, HAD-like effectors are also found in
other Legionella species suggesting that this
functional domain is widely used by these
pathogens for manipulation of host signaling
pathways. Despite commonality among HAD-
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Legionella phosphotyrosine phosphatase activity towards MAPKs
8
like effectors, Ceg4 represents a sequence-
distinct group containing eight Legionella
effectors that feature the combination of an N-
terminal HAD-like domain and a C-terminal
region carrying two transmembrane helices. The
C-terminal region of the Ceg4 effector plays an
important part in interactions of this effector with
eukaryotic MAP kinases as deletion of this region
had a significant effect on the ability of this
effector to dampen the signaling by the yeast
Fus3p MAPK. Furthermore, alanine substitution
of catalytic residues D11 and D158 in the Ceg4
HAD-like domain active site abrogated
phosphatase activity and ability to dampen
MAPK activation. Combined with the broad
substrate specificity of the Ceg4 HAD-like
domain toward phosphotyrosine peptides
demonstrated by our in vitro assays, this
observation prompted us to suggest that the C-
terminal region may be responsible for tailoring
the general phosphatase activity of Ceg4 toward
its specific host target.
The crystal structure of the Ceg4[1-208]
fragment showed strong similarity with
previously structurally-characterized members of
the HAD-like protein family including specific
features of the active site such as conservation of
key catalytic residues and coordination of a Mg2+
ion, known to be essential for catalysis (51).
Probing the Ceg4 active site cavity with site-
directed mutagenesis not only confirmed the role
of residues predicted to be directly involved in
catalysis but also revealed the subset of residues
important for Ceg4 activity against MAP kinase
substrates. Substitution of residues such as D11
which acts as a general acid/base, drastically
reduced or completely abrogated the activity of
this effector against Fus3 MAPK in a yeast model
system. Notably, previous prediction of specific
residues important for Ceg4 activity based on
similarity to Coxiella HAD-like effectors (43)
was only partially confirmed by our structure and
subsequent mutagenesis. Specifically, key
residues of motifs II and III were previously
predicted to correspond to Ser81 and Lys111.
However, our structural analysis pointed instead
to Thr103 and Lys135 fulfilling this role. This
observation highlights the limits of primary
sequence based analysis applied to highly diverse
HAD-like proteins, and effectors in general and
reiterates the necessity in molecular and
structural data to complement functional
diversity across large protein families.
The substrate specificity of HAD-like
domains is often defined by the ‘cap’ subdomain
insertion that controls the access to the catalytic
center (67). The Ceg4 HAD-like domain
structure features an unusual α-helical cap motif
never before described for this domain. This
novel cap motif is compatible with the broad
specificity of this domain against
phosphotyrosine peptides as indicated by our
observation that the tyrosine residue from two
different N-terminal tag sequences is able to
make intimate contacts with the Ceg4 active site
of a neighboring molecule in two different crystal
lattices. The specific position of the tyrosine
residue is the Ceg4 active site is compatible with
the position of trapped phosphate/phosphate
analog substrates in other structurally-
characterized HAD-like phosphatases,
suggesting that this crystallization observation
may indeed be representative of the interaction
between the Ceg4 HAD-like with its
phosphoprotein substrate.
Effectors have been demonstrated to
target all strata of the MAPK controlled signaling
pathways (MAPKKK, MAPKK, MAPK and
MKs) using several differing enzymatic activities
(68,69). Characterization of Ceg4 phosphatase
activity against yeast and human MAP kinases
adds a new member to this growing list of
MAPK-modulating factors along with the
recently-characterised Coxiella HAD-like
effectors active against the yeast CWI MAPK
(43). Human HEK293t cells transfected with
Ceg4 demonstrated clear reduction of the amount
of phosphorylated p38 MAP kinase, and this was
compromised by mutation of the key Ceg4 active
site residue D9. Previous work has shown that
MAPK phosphorylation in human cells is
increased at very early time points of bacterial
challenge by Legionella, and that this activation
is sustained for some time in an effector-
dependent manner (39). Therefore, it is tempting
to speculate that Ceg4 would serve a functional
role only at significantly later points of infection
and in keeping with this model, RNA-Seq data
taken during infection show that Ceg4
transcription levels are low during the post
exponential/infectious stage but increase 10-fold
during exponential growth (70). An additional
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Legionella phosphotyrosine phosphatase activity towards MAPKs
9
possibility is that given the C-terminal dependent
localization to the endoplasmic retuculum
membranes and the loss of ability of Ceg4 to
modulate Fus3 activation without this domain it
is possible that Ceg4 acts to reduce specific
MAPK activation in a localized manner, while
allowing general activation throughout the rest of
the cell.
Characterization of the structural and
functional features of the Ceg4 effector presented
in this work provide the first insight into function
of this and other HAD-like effectors during
Legionella infection, paving the way to further
studies into this bacteria’s pathogenic strategy.
EXPERIMENTAL PROCEDURES
Protein purification and size exclusion
chromatography-Based on domain and
transmembrane location predictions, genes
corresponding to Ceg4 residues 1-208 and 1-193
were cloned into p15Tv-LIC-TEV by ligation
independent cloning, and transformed into E. coli
BL21 CodonPlus (DE3) RIPL competent cells
using standard procedures. Several mutants of
Ceg4[1-193] in p15Tv-LIC-TEV (D9A, D11N,
D11A, E26A, D157A, D158A and D162A) along
with a variant of Ceg4[1-208] with the TEV
sequence ‘GRQNLYFQG’ mutated to match the
activation loop sequence from S. cerevisiae Hog1
‘PQMTGYVST’ were prepared by site-directed
mutagenesis. Cultures were grown at 37 °C in M9
media with selenomethionine or LB media,
supplemented with kanamycin and at an OD600
of 0.6-0.8 and expression of 6xHis-TEV tagged
Ceg4 was induced by the addition of 0.4 mM
isopropyl-β-D-1-thiogalactopyranoside and the
temperature of the culture was reduced to 16°C
overnight. The following day, cultures were
harvested by centrifugation and pellets lysed by
sonication on ice in 50 mM HEPES (pH 7.5), 150
mM NaCl, and 1 mM PMSF. All further
purification was conducted at 4 °C. Cell lysate
was clarified by ultracentrifugation at 17,000 × g
for 30 min, and 1–5 ml of Ni-NTA resin (Qiagen)
was added and incubated with gentle rotation for
30 min. Resin was washed with 50 mM HEPES
(pH 7.5), 150 mM NaCl, and 30 mM imidazole,
and protein eluted with 50 mM HEPES (pH 7.5),
150 mM NaCl, and 500 mM imidazole. Proteins
were further concentrated using a centrifugal
concentrator, flash-frozen in liquid N2, and
stored at −80 °C. Oligomerization of Ceg4[1-
208] was tested by size exclusion
chromatography using a Superdex S200 column
with running buffer 50 mM HEPES (pH 7.5) and
150 mM NaCl.
General enzyme activity screening–
General screens for enzyme activity were
performed as previously described (58) using
Ceg4[1-193]. Briefly, 20 µL of purified protein
(at 0.5 µg/µL) was added to wells of a 96-well
plate, then 180 µL of protease, phosphatase,
phosphodiesterase, dehydrogenase, oxidase,
NADH/NADPH oxidase and lipase, or 170 µL of
thioesterase mixes (containing buffers, metal
cations and substrates) were added, as well as 10
µL of 5,5'-dithiobis(2-nitrobenzoic acid) to the
thoiesterase mix. Plates were incubated at 37°C
for 1 hour. Phosphatase, phosphodiesterase,
protease, lipase, and thioesterase results were
read at 410 nm, dehydrogenase, oxidase, and
NADH/NADPH oxidase results were monitored
at 340 nm. All results were obtained using a
Spectramax M2 plate reader.
Natural phosphatase substrate
screening–Screens for activity towards naturally
occurring phosphatase substrates were performed
as previously described (58) using Ceg4[1-193].
Briefly, two 96-well plates were prepared, one as
a blank control and a second for the protein. 10
µL of natural phosphatase substrate at 4 mM (see
table S1 for list) was added to each assay well,
followed by 150 µL of reaction mixture or
reaction mixture containing 2 ug protein to give a
final concentration of 50 mM HEPES-K (pH 7.5),
5 mM MgCl2 1 mM MnCl2, and 0.5 mM NiCl2.
Plates were incubated at 37 °C for 30 mins. After
incubation, 40 µl malachite green development
reagent was added to each well prior to reading
the absorbance at 630 nm. (mM phosphate.min-
1.mg-1) was calculated based on a KH2PO4
standard curve.
Ceg4 metal and pH dependence assays
and kinetics using p-nitrophenylphosphate -
Assays for metal requirements were conducted
using 20 mM p-nitrophenylphosphate (pNPP) in
50 mM HEPES-K (pH 7.0), 0.1 µg.mL-1 of
purified Ceg4 [1-193] and the concentration of
metals indicated, in a final volume of 200 µL. For
pH optimizations, reactions were conducted
using 20 mM pNPP, 15 mM MgCl2, and 0.1
µg.mL-1 Ceg4 [1-193], and one of the following
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Legionella phosphotyrosine phosphatase activity towards MAPKs
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buffers at 50 mM; MES (pH 6.5)/HEPES (pH
7)/HEPES (pH 7.5)/HEPES (pH 8)/CHES (pH
9)/CHES (pH 9.5)/CHES (pH 10). For kinetics
determinations, pNPP was used at the
concentrations specified. Reactions were started
by the addition of enzyme and monitored using a
Spectramax M2 plate reader at 405 nm for 25
minutes at 25 °C. Data analysis and curve –fitting
was performed using Graphpad Prism 6
Phosphopeptide phosphatase assay–
Phosphatase activity towards a library of 53
phosphopeptide sequences was tested as
previously described (46). Briefly, 10 µL peptide
solution was mixed with 150 µL of 50 mM
HEPES (pH 7.0), 15 mM MgCl2 and 0.01 µg of
SBP-purified Ceg4[1-193]. The reaction was
incubated for 10 minutes at 25 °C before addition
of 40 µL of malachite green development reagent.
Absorbance at 630 nM was recorded. Enzyme
velocity (in mM phosphate.min-1.mg-1) was
calculated based on a KH2PO4 standard curve.
Subsequent kinetics determinations with peptide
QMTGpYVSTR were performed using the
method above, with the peptide concentrations
specified in the figure. Data analysis and curve –
fitting was performed using Graphpad Prism 6.
Crystallization, structure determination
and analysis–Crystals of selenomethionine-
substituted Ceg4[1-208]TEV site were grown at 23
°C using the hanging-drop vapor diffusion
method by mixing 2 μL of 102 mg/mL protein
with 2 μL of reservoir solution containing 0.2 M
MgCl2, 0.5 mM MnCl2, 0.1 M Tris pH 7.3, 30%
(w/v) PEG 4K and 2 mM phosphotyrosine.
Crystals of native Ceg4[1-208]HOG1p were grown
at 23 ◦C using hanging-drop vapor diffusion by
mixing 2 μL of 50 mg/mL protein with 2 μL of
reservoir solution containing 0.2 M NaCl, 0.1 M
Bis-Tris pH 6.5 and 25% (w/v) PEG 3350. All
crystals were cryo-protected with reservoir
solution supplemented with paratone oil.
Diffraction data were collected at 100 K
at beamline 19-ID at Structural Biology Center,
Advanced Photon Source at the wavelength of
0.979 Å (selenium peak). All diffraction data
were reduced with HKL-3000 (71). The Ceg4[1-
208]TEV site structure was solved first by SAD
phasing using PHENIX.solve (72) which
identified all five selenomethionine residues in
the asymmetric unit, followed by model building
by PHENIX.autobuild. The structure of Ceg4[1-
208]HOG1p was determined by Molecular
Replacement using the Ceg4[1-208]TEV site as
search model using PHENIX.phaser. All
structures were refined using PHENIX.refine and
Coot (73). The final Ceg4[1-208]TEV site model
includes the sequence GRQNLYFQG from the
TEV site followed by residues 1-204 of Ceg4; the
final Ceg4[1-208]HOG1p model includes the
sequence TGYVST from yeast Hog1 followed by
residues 1-204 of Ceg4, with residues 146 and
147 unmodeled due to poor electron density. B-
factors were refined as isotropic for all structures.
All geometries were verified with
PHENIX.refine and the wwPDB Validation
server. Structure coordinates were deposited to
the Protein Databank under accession codes
6AOK and 6AOJ for the Ceg4[1-208]TEV site and
Ceg4[1-208]HOG1p structures, respectively.
Structural orthologs were identified using the
Dali lite server (74). Active site volume was
calculated by the CastP (75)
Yeast transformation and MAPK
pathway activation assay–Ceg4[1-397] or the
mutants specified were cloned into pYES2 NT/A,
transformed into S. cerevisiae (W303 MATa,
bar1::NatR, far1Δ, mfa2::pFus1-GFP, ura3::Kan-
pSTL1-BFP, his3, trp1, leu2) using the LiAc
procedure (76) and grown on selective media for
two days at 30 °C. For analysis of mating and
high osmolarity glycerol (HOG) pathway
responses by flow cytometry, transformants were
grown either in duplicate or triplicate in selective
medium overnight at 30 °C. Empty plasmid was
grown as a negative control. Overnight cultures
were diluted to OD600 between 0.1-0.2 and
grown to early log phase. For analysis of mating
pathway response, yeast cells were treated with 1
mM α-factor and incubated at 30 °C for two
hours. For analysis of the HOG pathway
response, cells were treated with 2 mM KCl and
incubated at 30 °C for one hour. For both
conditions, cells were then treated with the
protein synthesis inhibitor cycloheximide for 30
minutes. For each sample, 10,000 cells were
measured with a MACSQuant Vyb (Miltenyi
Biotech). Data shown are mean fluorescence
(GFP for mating response and BFP for HOG
response) and standard deviation of duplicates or
triplicates.
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Legionella phosphotyrosine phosphatase activity towards MAPKs
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HEK293t and HeLa cell culture and
transfection–For MAPK activation assays,
HEK293t cells were maintained in Dulbecco’s
Modified Eagle’s Medium (DMEM),
supplemented with 10 % FBS at 37 °C and 5 %
CO2, and grown to a confluence of approximately
70 % at the time of transfection. Cells were
transfected using Lipofectamine 3000 (Life
Technologies) according to the manufacturer’s
instructions, with endotoxin free pcDNA3-N-
Flag-LIC containing Ceg4[1-397] or Ceg4[1-
397]D9A.
For cell localization studies, HeLa cells
were grown on poly-L-lysine treated glass
coverslips, maintained in DMEM, supplemented
with 10 % FBS at 37 °C and 5 % CO2, and grown
to a confluence of approximately 70 % at time of
transfection. Cells were transfected with either
pEGFP-N1 containing Ceg4[1-397], Ceg4[1-
397]D9A, Ceg4[1-207] or Ceg4[208-397] using
Lipofectamine 3000 (Life Technologies)
according to the manufacturer’s instructions
followed by counterstaining with DAPI during
fixation. For co-localization studies, pEGFP-N1
Ceg4[1-397]D9A transfected cells were grown in
chambered coverslips and incubated with ER-
tracker and Lyso-tracker red dyes according to
the manufacturer’s instructions prior to fixation
and counterstaining with DAPI. Microscopy data
were collected using a Nikon TiE inverted
microscope and Nikon C2 confocal system, with
a 60X oil immersion lens.
MAPK activation and Immunoblotting–
24 hours post-transfection with Ceg4[1-397] or
Ceg4[1-397]D9A, 5 µg/ml anisomycin or 200 nM
TPA was added to the culture medium. After 30
minutes, cells were gently washed once with PBS
followed by lysis directly into SDS-PAGE
loading buffer. SDS-PAGE was performed with
the addition of 0.5% 2,2,2-trichloroethanol added
to the resolving portion of the gel. After
electrophoresis, gels were exposed to UV light
for 2 minutes and imaged with a GelDoc
(BioRad) to obtain loading controls prior to
western blotting. Proteins were transferred to
nitrocellulose using a Transblot Turbo (BioRad).
Membranes were blocked with blocking buffer
(5% (w/v) BSA in TBS with 0.1% Tween 20) for
1 hour, followed by incubation with either anti-
p38 (Cell Signalling Technology, #8690, 1:1000
dilution in blocking buffer) or anti phospho-p38
(Cell Signaling Technology, #4511, 1:1000
dilution in blocking buffer) overnight. After
washing, membranes were incubated in 5% non-
fat skim milk in TBS with 0.1% Tween 20
containing anti-rabbit HRP (Cell Signalling
Technology, #7074, 1:4000 dilution) for 1 hour.
Blots were developed with BioRad Clarity
Western plus reagent and imaged using a GelDoc
(BioRad) and visualized using ImageLab
(BioRad).
Visualization of total cellular tyrosine
phosphorylation by immunoblot in S. cerevisiae
and HEK293T.. BY4741 S. cerevisiae (MATa
his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 (77)) were
transformed with the indicated constructs in
pYES2NT/A using the LiAc procedure (76) and
grown on CM agar -uracil selective media for two
days at 30 °C. Colonies were picked and cultured
overnight in selective media supplemented with
2% (w/v) raffinose. In the morning, 3ODs of cells
were harvested by centrifugation, washed and
resuspended in 5 ml selective media
supplemented with 2 % (w/v) galactose and
incubated at 30 °C with shaking for 5 hrs.
Cultures were harvested by centrifugation and
cells were lysed using an alkaline/SDS lysis
procedure (78). HEK293T cells were cultured in
and transfected in 6 well plates as per procedures
described for MAPK activation assays. Cells
were washed with PBS and harvested directly
using 100 µl/well of SDS-PAGE loading buffer,
followed by brief sonication to reduce viscosity
and 5 minutes of heating at 95 °C. 20 µl of each
yeast lysate and 15 µl was loaded and separated
with 12 % SDS-PAGE gels supplemented with
0.5 % (v/v) 2,2,2-trichloro ethanol, followed
visualization of protein loading after exposure of
gels to UV light for 2-3 minutes. Protein was
transferred to nitrocellulose for immunoblotting
using a Transblot Turbo (BioRad). For total
phosphotyrosine detection, yeast and HEK293T
blots were blocked with 5% w/v BSA, 1X TBS,
0.1% Tween 20 at 4°C for 1hr with gentle shaking
followed by overnight incubation with α-P-Tyr-
100 antibody (Cell Signalling Technology,
#9411, 1:2000 in blocking buffer). For Ceg4
detection in yeast, blocking was performed in 5%
non-fat skim milk in TBS with 0.1% Tween 20
followed by incubation with α-Xpress
(Thermofisher Scientific, #R910-25 1:4000)
overnight. For detection of Ceg4 in HEK293T
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Legionella phosphotyrosine phosphatase activity towards MAPKs
12
cells, blocking was performed in 3% non-fat skim
milk in TBS followed by incubation with α‐
FLAG M2 (1:2000, Cat# F1804, Sigma‐Aldrich)
for 45 minutes at room temperature.
After 3, 5 minute washes with TBS with
0.1% Tween 20, all blots were incubated in 5%
non-fat skim milk in TBS with 0.1% Tween 20
containing anti-mouse HRP (Cell Signalling
Technology, #7076, 1:4000 dilution) for 1 hour.
Blots were developed with BioRad Clarity
Western plus reagent and imaged using a GelDoc
(BioRad) and visualized using ImageLab
(BioRad).
Acknowledgements: Special thanks to Greg Brown, for his invaluable assistance with screening and
activity assays. We would also like to thank the Claycomb lab for training and use of their microscopy
facilities.
Conflict of interest: The authors declare no conflicts of interest.
Author contributions: AQ performed human in vivo work with the help of DV. EE and OE purified and
crystalized Ceg4. OE and AQ performed enzyme activity screening. PK and SP performed yeast MAPK
assays. BN collected crystal data. PS solved the structures and wrote the manuscript. AQ and AS wrote the
manuscript with input from AWE and AS. All work was performed under the supervision of AWE, AFY
and AS.
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Legionella phosphotyrosine phosphatase activity towards MAPKs
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FOOTNOTES
Results shown in this report are supported in part by a National Institutes of Health grant (GM094585) to
AS through the Midwest Center for Structural Genomics and by the U. S. Department of Energy, Office of
Biological and Environmental Research (contract DE‐AC02‐06CH11357). The work performed at Argonne
National Laboratory, Structural Biology Center at the Advanced Photon Source. Argonne is operated by
UChicago Argonne, LLC, for the U.S. Department of Energy, Office of Biological and Environmental
Research under contract DE-AC02-06CH11357
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TABLES
Table 1 – X-ray diffraction data collection and refinement statistics
Structure
Ceg4[1-208]TEV site
Se-Met
Ceg4[1-208]HOG1p
Native
PDB Code
6AOK
6AOJ
Data collection
Space group
P212121
C2
Cell dimensions
a, b, c (Å)
β (°)
40.04, 48.03, 100.10
90.0
78.97, 75.92, 47.91
104.2
Resolution, Å
40.0 – 1.88
50.0 – 1.90
Rmergea
Rpim
0.071 (0.525)b
0.035 (0.266)
0.052 (0.259)
0.031 (0.149)
I / (I)
21.4 (3.9)
39.3 (4.5)
Completeness, %
100 (99.9)
97.2 (92.5)
Redundancy
5.1 (4.9)
3.9 (3.8)
Refinement
Resolution, Å
37.17 – 1.88
36.53 – 1.90
No. of unique reflections:
working, test
16264, 1461
20876, 1044
R-factor/free R-factorc
15.6/19.8 (19.9/27.1)
16.2/19.3 (22.1/28.3)
No. of refined atoms
Protein
Magnesium
Chloride
Water
1811
1
2
269
1738
1
1
252
B-factors
Protein
Magnesium
Chloride
Water
23.1
12.7
23.5
37.8
51.4
32.7
38.3
59.3
r.m.s.d.
Bond lengths, Å
Bond angles,
0.016
1.267
0.009
0.954
aRsym = hi|Ii(h) - I(h)/hiIi(h), where Ii(h) and I(h) are the ith and mean measurement of the intensity
of reflection h.
bFigures in parentheses indicate the values for the outer shells of the data.
cR = |Fpobs – Fpcalc|/Fpobs, where Fpobs and Fpcalc are the observed and calculated structure factor amplitudes,
respectively.
* = molecules in the active site cleft.
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Legionella phosphotyrosine phosphatase activity towards MAPKs
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FIGURE LEGENDS
FIGURE 1. Domain organization and species Ceg4 orthologues. Phylogenetic analysis of the HAD
domains of Legionella Dot/ICM effectors shows that Ceg4 (Lpg0096) belongs to a group of effectors in
which the majority of members possess a two transmembrane (TM) region. Numerals indicate node
posterior probabilities, scale bar illustrates substitutions per site.
FIGURE 2. Ceg4 is a phosphotyrosine phosphatase. A, General screening for a range of activities
indicated that Ceg4 is a phosphatase. B, Kinetics were obtained for the generic phosphatase substrate pNPP.
Error bars denote SEM values. C, Incubation of Ceg4 with a variety of naturally occurring phosphatase
substrates, and subsequent detection of released phosphates, reveals that Ceg4 preferentially removes the
phosphate from phosphotyrosine in vitro. See also supplemental data, Figure S1E for the full list of tested
substrates. Error bars show 95% confidence intervals from triplicate data. D, Kinetics were obtained for the
generic phosphatase substrate pNPP. Error bars denote SEM values E, Ceg4 dephosphorylates all tested of
phosphotyrosine-containing peptides at levels 4-5 times higher than phosphoserine or phosphothreonine-
containing peptides in vitro. Error bars denote 95% confidence intervals from triplicate data. See also
Supplemental Figure S1E for all of tested peptides.
FIGURE 3. Crystal structure of Ceg4’s HAD-like domain provides molecular insight into phosphatase
activity. A, Schematic of the final structural model of Ceg4. Amino acids highlighted in blue belong to the
core Rossmann-like fold, gray to the cap domain and orange to the expression tag used for purification. B,
Overall structure of the Ceg4 HAD-like domain, highlighting positions and topology of the C1-cap
subdomain and the ‘squiggle motif’.C, Close-up of the Ceg4 HAD-like domain active site, indicating
positions of catalytic site residues (small red spheres denote positions of water). D, Catalytic site residues
of Ceg4 form a ~350 Å pocket, occupied by a magnesium and a chloride ion and partially covered by N20
of the cap subdomain.
FIGURE 4. Ceg4[1-208] crystallizes with the N-terminal fusion tag’s tyrosine bound deeply in the catalytic
pocket of the adjacent molecule. A, Crystal packing of Ceg4[1-208]HOG1p shows the N-terminal peptide
packed against the active site of the adjacent Ceg4[1-208]HOG1p molecule. B, Close-up examination of the
HOG1p tag-Ceg4 interaction shows the tyrosine bound deeply in the catalytic pocket. C, Hog1 and TEV
fusion tag peptides bind the active site of Ceg4[1-208] with strikingly similar conformations.
FIGURE 5. Ceg4 supresses MAPK responses in S. cerevisiae. A, Ceg4[1-397] is able to reduce the Hog1-
mediated high osmolarity response to high salt concentrations and the Fus3 mediated pheromone/mating
response to α-factor. Removal of the C-terminal TM domain reduced Ceg4’s ability to lessen the pheromone
response, but slightly increased its ability to reduce activation of the hyperosmolarity response, suggesting
off-target effects. B, Ceg4[1-397] mutants K17A, N20A, E26A, E108A, E131A and T161A all retained
wildtype levels of activity, whereas mutants D11A, V23D, F24A, Y43A, K104A, K135, and D158 all
exhibited reduced ability to supress the pheromone response. See also Supplementary Figures S5 for
expression testing. C, Location of tested mutants illustrated on the structure of Ceg4. Mutation of residues
highlighted in red reduced ability to supress the pheromone response.
FIGURE 6. Ceg4 localization to the endoplasmic reticulum requires C-terminal TM domain and dampens
activation of human MAPK p38 in vivo. A, Ceg4 localization is dependent on C-terminal TM domains. N-
terminal GFP fusions of Ceg4[1-397], Ceg4[1-397]D9A and Ceg4[208-397] expressed in HeLa cells show
distinct subcellular localisation that is lost in Ceg4[1-207] lacking transmembrane regions. B, GFP-Ceg4[1-
397]D9A colocalizes with ER-tracker red dye. See also Supplemental figure S6. C, Activation and
phosphorylation of p38 MAPK was achieved by incubating HEK293 cells expressing Ceg4[1-397] or
Ceg4[1-397]D9A with TPA or anisomycin. While total levels of p38 remained unchanged, cells expressing
Ceg4[1-397] showed reduced levels of phospho-p38 compared to D9A mutant constructs.
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Alexander W. Ensminger and Alexei Savchenko
Boguslaw Nocek, Purnima S. Kompella, Sergio Peisajovich, Alexander F. Yakunin,
Andrew T. Quaile, Peter J. Stogios, Olga Egorova, Elena Evdokimova, Dylan Valleau,
attenuates activation of eukaryotic MAPK pathways
The Legionella pneumophila effector Ceg4 is a phosphotyrosine phosphatase that
published online January 4, 2018J. Biol. Chem.
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