Dimerization of Vaccinia virus VH1 is essential for dephosphorylation of STAT1 at tyrosine 701.

Abstract

The gene product of Vaccinia virus gene H1, VH1, is the first identified dual specificity phosphatase (DSP). The human genome encodes 38 different VH1-like DSPs, which include major regulators of signaling pathways, highly dysregulated in disease states. VH1 down-regulates cellular antiviral response by dephosphorylating activated STAT1 in the IFN-γ/STAT1 signaling pathway. In this report, we have investigated the molecular basis for VH1 catalytic activity. Using small-angle x-ray scattering (SAXS), we determined that VH1 exists in solution as a boomerang-shaped dimer. Targeted alanine mutations in the dimerization domain (aa 1-27) decrease phosphatase activity while leaving the dimer intact. Deletion of the N-terminal dimer swapped helix (aa 1-20) completely abolishes dimerization and severely reduces phosphatase activity. An engineered chimera of VH1 that contains only one active site retains wild-type levels of catalytic activity. Thus, a dimeric quaternary structure, as opposed to two cooperative active sites within the same dimer is essential for VH1 catalytic activity. Together with laforin, VH1 is the second DSP reported in literature for which dimerization via an N-terminal dimerization domain is necessary for optimal catalytic activity. We propose that dimerization may represent a common mechanism to regulate the activity and substrate recognition of DSPs, often assumed to function as monomers.

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Dimerization of Vaccinia Virus VH1 Is Essential for
Dephosphorylation of STAT1 at Tyrosine 701*
Receivedforpublication,January28,2011,andinrevisedform,March1,2011 Published,JBCPapersinPress,March1,2011,DOI10.1074/jbc.M111.226357
Adem C. Koksal‡§and Gino Cingolani‡1
Fromthe‡DepartmentofBiochemistryandMolecularBiology,ThomasJeffersonUniversity,Philadelphia,Pennsylvania19107and
the§DepartmentofBiochemistryandMolecularBiology,SUNYUpstateMedicalUniversity,Syracuse,New York 13210
The gene product of Vaccinia virus gene H1, VH1, is the first
identified dual specificity phosphatase (DSP). The human
genome encodes 38 different VH1-like DSPs, which include
major regulators of signaling pathways, highly dysregulated in
disease states. VH1 down-regulates cellular antiviral response
by dephosphorylating activated STAT1 in the IFN-?/STAT1
signaling pathway. In this report, we have investigated the
molecular basis for VH1 catalytic activity. Using small-angle
x-rayscattering(SAXS),wedeterminedthatVH1existsinsolu-
tionasaboomerang-shapeddimer.Targetedalaninemutations
in the dimerization domain (aa 1–27) decrease phosphatase
activity while leaving the dimer intact. Deletion of the N-termi-
nal dimer swapped helix (aa 1–20) completely abolishes
dimerization and severely reduces phosphatase activity. An
engineered chimera of VH1 that contains only one active site
retainswild-typelevelsofcatalyticactivity.Thus,adimericqua-
ternary structure, as opposed to two cooperative active sites
within the same dimer is essential for VH1 catalytic activity.
Together with laforin, VH1 is the second DSP reported in liter-
ature for which dimerization via an N-terminal dimerization
domain is necessary for optimal catalytic activity. We propose
that dimerization may represent a common mechanism to reg-
ulate the activity and substrate recognition of DSPs, often
assumed to function as monomers.
Dual-specificity phosphatases (DSPs)2represent a subclass
oftheprotein-tyrosinephosphatase(PTP)superfamilythatcan
dephosphorylate both phosphotyrosine and phosphoserine/
threoninecontainingsubstrates(1–5).ThefirstidentifiedDSP,
VH1, is encoded by the conserved H1 locus of Vaccinia virus
(6). In the past 20 years the list of identified VH1-like DSPs has
been greatly expanded and now includes 61 different members
(5). Based on sequence similarity and presence of functional/
binding domains, DSPs are usually divided into 7 diverse sub-
groups (5).
Like classical PTPs, VH1-like DSPs contain a catalytic triad
consisting of a cysteine, an arginine, and an aspartic acid, usu-
allyarrangedinthecontextofanextendedconsensusmotif(7).
DSPsemployasimilarcatalyticmechanismasPTPs,character-
ized by the formation of a transient enzyme-phosphosubstrate
intermediate (1, 2). Similar to PTPs, the DSP catalytic core
shows a great degree of substrate specificity. The substrate
however, can be non-peptidic for a number of DSPs. For
instance, PTEN-like phosphatases dephosphorylate D3-phos-
phorylated inositol phospholipids (8), or the DSPs PIR (also
knownasDUSP11)andlaforinhavebeenshowntodephosphor-
ylate mRNA (9) and phosphoglucans (10), respectively.
The gene encoding VH1 is highly conserved among double-
stranded DNA viruses of the Poxviridae family (11). Approxi-
mately200moleculesofVH1arepackagedwithintheVaccinia
virionandareessentialfortheviabilityofVacciniavirus(12).In
vivo, VH1 is required for maturation of two-virion membrane-
associated factors, namely A17 (13) and A14 (14). Upon infec-
tion, the DSP is released into the host cytoplasm, where Vac-
cinia virus establishes cell factories and replicates (15).
Growing evidence indicates that VH1 blocks the host interfer-
on-?(IFN-?)signaltransductionpathwaysbydephosphorylat-
ing the transcription factor STAT1 at tyrosine 701 (Tyr-701)
(16). This was demonstrated using both activated STAT1
immunoprecipitated from cells stimulated with IFN-? (16), as
wellasrecombinantSTAT1invitrophosphorylatedatTyr-701
(17). Notably, VH1 phosphatase activity is specific to STAT1
andSTAT2,butnotSTAT3andSTAT5(18).ActivatedSTAT1
is imported into the nucleus by the transport adaptor importin
?5boundtoimportin?(19).BindingofSTAT1toimportin?5
requires tyrosine phosphorylation at position 701, which is the
specificsubstrateofVH1.Invitro,importin?5protectsSTAT1
from VH1-mediated dephosphorylation in a dose-dependent
manner (17). Because importin ?5 does not bind pTyr701 in
STAT1 (20), the decreased susceptibility to VH1 in the pres-
enceofimportin?5islikelycausedbythestructuralconstraints
imposed by importin ?5 binding to STAT1. The competition
with importin ?5 together with the observation that VH1 is
inactive with respect to DNA-bound STAT1 suggested that
VH1 acts predominately on the cytoplasmic pool of activated
STAT1 (17). Thus, VH1-mediated blockage of the IFN-?/
STAT1 signaling pathway has the likely function to prevent
transcription of downstream STAT1 target genes, and thereby
block or reduce an antiviral response.
The crystal structure of Vaccinia (17) and its closely related
Variola (11) virus VH1 phosphatases were recently deter-
mined. In both cases, the structure revealed a conserved PTP-
* Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealth
Grant GM074846-01A1.
1To whom correspondence should be addressed: Dept. of Biochemistry and
Molecular Biology, Thomas Jefferson University, 233 South 10th Street,
Philadelphia,PA19107.Tel.:215-503-4573;Fax:215-923-2117;E-mail:gino.
cingolani@jefferson.edu.
2Theabbreviationsusedare:DSP,dualspecificityphosphatase;PTP,protein-
tyrosine phosphatase; OMFP, 3-O-methylfluorescein phosphate; IFN-?,
interferon-?; CD, circular dichroism; STAT, signal transducer and activator
of transcription; pTyr701, phosphotyrosine 701; anti-pTyr, anti-phospho-
tyrosine; SAXS, small angle x-ray scattering; NSD, normalized spatial dis-
crepancy; mVH1, monomeric VH1; dVH1, dimeric VH1; chVH1, chimeric
VH1; aa, amino acids.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 16, pp. 14373–14382, April 22, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in the U.S.A.
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like fold similar to that previously reported for VHZ (21) and
VHR (22). The VH1 active site consists of a shallow catalytic
cleft only ?6 Å deep, which can accommodate both phospho-
tyrosineandphosphothreonine/serineresidues.Thisactivesite
structurecontrastsfromclassicaltyrosinephosphatases,where
the catalytic cysteine is located at the bottom of a ?9 Å deep
pocket,highlyselectivetorecognizephosphotyrosine(22).Fur-
thermore, in both Vaccinia and Variola DSPs, a putative
dimeric quaternary structure was inferred by analysis of crystal
contacts (11, 17). Dimerization of VH1 has not been validated
in vivo and its putative physiological significance remains
unclear. Intriguingly, the main cellular substrate of VH1, phos-
phorylated STAT1 (pSTAT1), is also dimeric upon phosphor-
ylation at Tyr-701 (23). Therefore, it was proposed that the
VH1 dimeric quaternary structure represents an evolutionary
adaptation to pSTAT1 specific recognition (17).
In the present study, we have analyzed the mechanisms by
which dimerization affects VH1 catalytic properties. We found
that while there is no cross-talk between VH1 active sites, the
integrity of the VH1 dimeric quaternary structure is essential
for its catalytic activity and recognition of pSTAT1.
EXPERIMENTAL PROCEDURES
BiochemicalTechniques—Thegene-encodingVacciniavirus
VH1 was cloned in a pET-14b vector (Novagen) containing a
PreScission Protease cleavage site between the His6tag and the
first residue of VH1. VH1 was expressed in the Escherichia coli
BL21(DE3)strainandpurifiedaspreviouslydescribed(17).The
His6tag was cleaved off by incubating with PreScission Prote-
ase,followedbygelfiltrationchromatographyonaSuperdex75
column (GE Healthcare Life Sciences) in GF buffer (150 mM
sodium chloride, 20 mM HEPES pH 7.0, 3 mM ?-mercaptoeth-
anol, 2% glycerol, 0.1 mM PMSF). Monomeric VH1 lacking the
N-terminal residues 1–20 was generated by deletion PCR. The
proteinwasexpressedandpurifiedaswild-type(wt)VH1.Chi-
meric VH1 constructs were generated by introducing a 18
nucleotide linker (GGCGGAGGTGGCGGATCC) between
two VH1 genes. Point mutations in the active sites were intro-
duced by site-directed mutagenesis. All chimeras were ex-
pressed and purified as wt VH1. Human STAT1 was expressed
and phosphorylated at position 701 in E. coli strain TKX-1
(Stratagene, La Jolla, CA), which harbors an inducible plasmid
encoding a tyrosine kinase gene (pTK). Expression, in vivo
phosphorylation and purification of STAT1 were performed as
previously described (17).
SAXS Data Collection, Analysis, and ab Initio Shape
Reconstruction—Small-angle x-ray scattering data were mea-
sured at beam line X9A at the National Synchrotron Light
Source (Upton, NY). Data were collected by passing VH1 sam-
plesthroughaflowcapillaryatarateof10?l/min.Thesamples
were in the GF buffer previously mentioned, and at concentra-
tions ranging from 1–5 mg/ml. They were centrifuged at
16,000 ? g for 10 min before 20-s exposures were taken in
triplicates. The GF buffer was used in blank experiments to
subtractthesolventfromVH1samplescattering.Guinieranal-
ysis between the triplicate exposures was used to control for
radiation damage and signs of aggregation. Data reduction was
done by circular averaging of the images and scaling to obtain
the scattering curve (scattering intensity (I) as a function of the
momentumtransfervectorq(q?4?(sin?)/?).GNOM(24)was
used to calculate P(r) plots from the scattering data. Ab initio
modelcalculationstogenerateascatteringenvelopeweredone
using GASBOR (25). 14 solutions from GASBOR were used to
check consistency and averaged to obtain the final model using
the DAMAVER program suite (26). The final solution model
was converted to a surface map using the SITUS program suite
(27). The theoretical solution scattering of the crystal structure
of VH1 was calculated using CRYSOL (28). All figures in the
report were prepared using the program Pymol (29).
Sedimentation Velocity and Thermal Stability—Analytical
ultracentrifugation experiments for dimeric, monomeric, and
chimeric VH1 were carried out in 20 mM HEPES pH 7.0, 150
mM sodium chloride in a Beckman XL-A Analytical Ultracen-
trifuge (AUC) under velocity sedimentation mode. 450 ?l of
sample and 400 ?l of reference buffers were loaded into sepa-
rate compartments of a 12 mm path-length Epon centerpiece
cell.Runswereperformedat45,000rpmand10 °C.Absorbance
valueswerecollectedatawavelengthof276nmusingaprotein
concentration of 50 ?M. The data were fit to a continuous sed-
imentation coefficient (c(s)) distribution model and an esti-
mated molecular mass was obtained with the program SEDFIT
(30, 31). Thermal stability assays were recorded using a Jasco
J-810 spectropolarimeter equipped with a Neslab RTE7 refrig-
erated recirculator. VH1 samples at a final concentration of 7.0
?m in TM buffer (20 mM sodium phosphate (pH 7.4) and 100
mM NaCl) were measured using a 1 mm rectangular quartz
cuvette (Starna Cells, Inc.). The variations in ellipticity at 220
nm as a function of temperature in 1 °C increments were mea-
sured over the range 30 °C-75 °C. Slow cooling to 25 °C fol-
lowed by a CD scan for secondary structure demonstrated that
the unfolding of all VH1 samples is irreversible.
Phosphatase Kinetic Assays—The assay was performed in
dephosphorylation buffer (50 mM HEPES pH 6.8, 50 mM NaCl,
and 10 mM ?-mercaptoethanol). The enzymatic activity of the
various VH1 constructs was monitored using the colorimetric
substrate 3-O-methylfluorescein phosphate (OMFP, Sigma-
Aldrich) at an absorbance of 477 nm using varying concentra-
tions of substrate ranging from 0.1 to 10 times the Kmvalues.
The reaction was initiated by addition of enzyme at a final
dimer concentration of 1 ?M to the reaction mix at 37 °C. All
experiments were done in triplicate to calculate standard devi-
ations.Km,Vmax,andkcatvaluesweredeterminedbynon-linear
regression using the software GraphPad (GraphPad Software,
Inc).
STAT1 Dephosphorylation Assay—In vivo phosphorylated
STAT1 was used at a concentration of 1 mg/ml. All VH1 sam-
ples were added at a final concentration of 24 ?M. The final
reactionconditionswere50mMHEPESpH7.0,70mMNaCl,10
mM ?-mercaptoethanol in a volume of 50 ?l at 37 °C. Time
points were taken at 0, 1, 5, 30, 60, and 120 min. STAT1 phos-
phorylation levels were measured by Western blot analysis
using p-701-STAT1 (Cell Signaling Technology, Inc.) rabbit
primary mAb and HRP-goat anti-rabbit secondary antibody
(Invitrogen). All dephosphorylation reactions were repeated a
minimum of three times. The software ImageJ (NIH) was used
VH1CatalyticActivitythroughDimerization
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toquantifyrelativeintensitiesofpTyr701andplottedusingthe
program SigmaPlot.
RESULTS
Dimeric Structure of VH1 in Solution Investigated by Small-
Angle X-ray Scattering (SAXS)—The crystal structure of Vac-
cinia(17)andVariola(11)virusVH1wererecentlyreported.In
both structures, the crystallographic asymmetric unit contains
a single DSP-monomer. However, a dimeric quaternary struc-
turewasproposedbasedonanalysisofcrystalcontacts.Inboth
structures, applying 2-fold crystallographic symmetry gener-
ates a dimeric structure, which is stabilized by an N-terminal
domain swap of the first ?27 amino acids (11, 17). Although
Vaccinia virus VH1 dimerization was also confirmed in solu-
tionusinganalyticalultracentrifugationsedimentationvelocity
analysis (17), it is unclear if the quaternary structure proposed
using crystallographic symmetry represents the actual dimer
assembled in solution. To answer this question, we determined
the structure of VH1 in solution using SAXS. We measured
smallandwideanglescatteringdataforVH1atseveralconcen-
trations and obtained similar results in each case (Fig. 1A). The
gyration radius (Rg) and maximum dimension (Dmax) calcu-
lated from the experimental scattering are 26.3 Å and 87 Å,
respectively. This agrees well with the calculated Rg? 26.1 Å
FIGURE1.DimericquaternarystructureofVH1determinedinsolutionbySAXSanalysis.A,rawexperimentalscatteringdataforVH1(red)overlaidtothe
scattering curve calculated from the dimeric crystal structure (PDB 3CM3) (17) (black). The log of scattering intensity I(q) is plotted as a function of the
momentumtransfervectorq.ThedimericcrystalstructureofVH1wasgeneratedbyapplying2-foldcrystallographicsymmetrytotheasymmetricunitcontent
of VH1 crystal structure reported by Koksal et al. (17). B, distance distribution function P(r) plotted against the interatomic distance (r) for experimental SAXS
data (red) and data calculated from the crystal structure (black). The distribution for both data sets shows excellent agreement between SAXS data and the
dimericcrystalstructureofdVH1,withanapproximateRmaxat90Å.C,finalSAXS-envelopeofVH1calculatedfromexperimentalscatteringvalues.D,fittingthe
crystalstructureofdVH1withtheSAXS-envelopedemonstratesexcellentagreementbetweenthetwomodels.InbothpanelsCandD,theSAXSenvelopeon
the left is calculated without imposing symmetry; on the right is the 2-fold averaged SAXS-envelope.
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andDmax?90ÅfordimericVH1observedcrystallographically
(CRYSOL chi value ?2.4) (28). We generated a shape recon-
struction from the scattering data using the program GASBOR
(25), as described under “Experimental Procedures.” As shown
inFig.1C,theaveragedSAXSenvelopeforVH1obtainedhasan
elongatedboomerangshape.Thisenvelopefitswellthecrystal-
lographic structure of dimeric VH1, which has a slightly elon-
gated shape of ?85 Å in length, ?31 Å in height and ?28 Å in
width (Fig. 1D) (17). Ab initio reconstructions of 14 models
from 14 independent GASBOR iterations all agreed exception-
allywellwithoneanotherwithanormalizedspatialdiscrepancy
value of ?0.8 (this value defines the similarity between two
intrinsically distinct models, where a value smaller than 1.0
indicates a high degree of agreement between the two struc-
tures).Applying2-foldsymmetrytotheSAXSenvelopefurther
improved the quality of the VH1 reconstruction, which best
demonstrates the correctness of our solution (Fig. 1, C and D).
TheoreticalscatteringprofilesforthedimericstructureofVH1
weregeneratedandareingoodagreementwiththeexperimen-
tal data (Fig. 1A). Likewise, the agreement between VH1
dimeric crystal structure and SAXS data is also mirrored by
comparing experimental and calculated the P(r) shape func-
tions (Fig. 1B), which represent a distribution of interatomic
distances present in the molecule. The match between calcu-
lated and experimental P(r) functions for VH1 is remarkably
good, which confirms the crystallographic dimer built using
2-fold crystallographic symmetry indeed corresponds to the
conformation of VH1 adopted in solution. The shape function
associated with the crystallographic model of VH1 is only
slightly compressed toward an interatomic distance of ?40 Å,
which corresponds to the distance between two VH1 active
sites in the crystallographic dimer (Fig. 1D). This is likely
explained by the breathing of VH1 dimer in solution, which is
notseeninthecrystalstructure.Withtheonlyexceptionofthis
deviation, the dimeric crystal structure of VH1 previously
reported fits exceptionally well with the SAXS envelope, sug-
gestingthattheVH1domain-swappeddimerizationinterfaceis
not an artifact of crystallization.
Residues 1–27 of VH1 Form a Dimerization Domain Highly
Conserved in Poxviridae—As seen in the crystallographic
model (17), VH1 dimerization is mediated by swapping the
N-terminal residues 1–27, of which the first 17 residues fold
into a straight ?-helix (?1) (Fig. 2, A and B). Within a dimer of
VH1, the two N-terminal swapped helices, ?1A and ?1B, cross
each other at an angle of ?95° and make approximately fifteen
specific side chain contacts (17). The complexity of this
dimerization interface prompted us to analyze the amino acid
conservation of VH1 both in the DSP catalytic core and the
N-terminaldimerizationregion(Fig.2A).BLASTanalysisofall
VH1-like DSP sequences in the protein database indicates that
residues 1–27 are present only within virally encoded DSPs. In
particular, comparison of the VH1 gene in 37 Poxviridae
genomes reveals that residues 1–27 are remarkably well con-
served (80% identity), even in viruses that infect highly distinct
hosts. In contrast, VH1 DSP core (aa 28–171) is moderately
conserved (?30% amino acid identity) both within viral and
non-viral VH1-like DSPs. Despite the poor sequence conserva-
tion, structurally, VH1-DSP core superimposes accurately to
the DSP catalytic core of VHZ (21) (Fig. 2C) and of other DSPs
inthestructuraldatabase(datanotshown).Thus,thelongcon-
sidered prototypical member of the DSP-subfamily of PTPs,
VH1, presents a more complex structure than a minimal DSP
core,anditisfoundonlyinvirusesofthePoxviridaefamily.We
willrefertoVH1asaC-terminalDSPcore(aa28–171)fusedto
anN-terminaldimerizationdomain(DD,aa1–27)formedbya
helix-loop motif (Fig. 2A).
Point Mutations at VH1 Dimerization Interface Reduce Cat-
alytic Activity—Having established that the VH1-DD is not
conserved in other DSPs outside Poxviridae, we asked whether
thisdomainisinfactrequired,orcanmodulate,VH1phospha-
tase activity. Given that the DD is not part of the minimal cat-
alytic core, we set out to answer this question by introducing
alanine point mutations in key residues of the N-terminal
swapped helix (Fig. 2A). First, we mutated Ser-14 and Thr-15
(mutant dVH1–2m) that mediate packing of the ?1 helix
againsttheDSPcore(Fig.2B).Then,weintroducedthreemore
mutations in residues protruding on the surface of swapped
helix: Lys-8, Leu-11, and Leu-12 (mutant dVH1–5m) (Fig. 2, A
and B). The two mutants were expressed and purified under
identical conditions as wild-type VH1 (henceforth referred to
as dVH1). By sedimentation velocity analysis, both VH1 point
mutants migrated as monodisperse dimers ?40 kDa in molec-
ular mass, indistinguishable from dVH1 (Table 1). Therefore,
even5pointmutationsinVH1-DDwereinsufficienttodisrupt
the phosphatase dimeric structure. To analyze the effect of
these mutations on VH1 activity, we carried out an in vitro
dephosphorylation assays using OMFP. Interestingly, both
dVH1–2m and dVH1–5m hydrolyzed OMFP with reduced
efficiency as compared with the wt enzyme. The kcat/Kmvalues
for these two mutants were ?1.7- and 2.5-fold lower than that
of dVH1 (Table 1). The observed loss in efficiency was mainly
due to a decrease in affinity for the phosphosubstrate as wit-
nessed by a 3- and 4-fold increased Kmfor dVH1–2m and
dVH1–5m, respectively, as compared with dVH1 (Table 1).
Because both mutants retain a dimeric structure, it is plausible
that mutations in VH1-DD weaken the compactness of this
binding interface that is likely required for optimal catalytic
activity.
Monomeric VH1 Is Poorly Active—To determine the struc-
tural role of dimerization on catalytic activity, we investigated
the activity of a monomeric VH1 (Fig. 2, A and C). Given that
five point mutations in the DD were not sufficient to disrupt
VH1 dimerization, we deleted the N-terminal helix ?1 (aa
1–20), which is the main structural determinant bridging two
VH1-DSP cores together in a functional dimer (Fig. 2B). This
construct, mVH1, corresponds to VH1 minimal DSP core and
is predicted to fold into a discrete VHZ-like core (21) ?21 kDa
inmolecularmass(Fig.2C).mVH1wasexpressedinE. coliand
purifiedfromasolublefractionusinganN-terminalHistag.To
confirmmVH1ismonomericandcorrectlyfolded,wecarriedout
hydrodynamicandfoldingstudies.Bysedimentationvelocityanal-
ysis,mVH1migratedasamonodispersespecies?22kDa(Fig.3A,
Table 1), in agreement with the expected mass of ?21 kDa. Fur-
thermore,thenormalizedmolarellipticityformVH1showedmin-
imal loss of ?-helical signal as compared with the dimeric phos-
phatase (data not shown), consistent with a well folded DSP core.
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To gain insight into mVH1 structural stability, we recorded tem-
perature-mediated unfolding curves for mVH1 and dVH1, by
measuringvariationsinellipticityat220nmasafunctionoftem-
perature(Fig.3B).Interestingly,mVH1displayedabroadunfold-
ing transition with an apparent melting temperature (Tm) of
?52°C.AlthoughtheTmfordimericandmonomerVH1arevery
similar (?55°C versus 52°C) (Fig. 3B), the two proteins denature
in drastically different ways. dVH1 thermal unfolding is highly
cooperative and can be interpreted as a dimer of folded VH1
unfoldingtoamonomer.Incontrast,mVH1unfoldsnon-cooper-
atively,asoftenseenforsmallglobularmonomericproteins,char-
acterized by formation of unfolding intermediates en route to the
final unfolded state (32). Taken together, these data suggest that
deletingtheN-terminalswappedhelix?1inVH1-DDdisruptsthe
enzyme dimeric quaternary structure without affecting the DSP
coretertiarystructure.
To analyze mVH1 activity, we carried out an in vitro dephos-
phorylationassayusingOMFP.WefoundthatwhiledVH1hydro-
lyzesOMFPefficiently(Kmof87.17?Mandkcat/Km?2.05?106,
Table 1), mVH1 has dramatically lower affinity for OMFP (Km
?780 ?M) and ?120-fold reduced catalytic efficiency (kcat/Km?
0.017?106),ascomparedwiththedimericenzyme(Table1).The
rate of OMFP dephosphorylation observed for mVH1 is higher
thanthespontaneousrateofOMFPhydrolysisinanaqueousenvi-
ronment and follows Michaelis-Menten saturation kinetics (Fig.
3C). The monomeric phosphatase is, however, only minimally
active. Thus, a dimeric quaternary structure and not simply a
foldedDSPcoreisessentialforVH1optimalcatalyticactivity.
FIGURE2.VH1containsanN-terminaldimerizationdomainfusedtoaC-terminalDSPcore.A,schematicdiagramofaprotomerofVH1withDDdomain
and DSP core colored in red and green, respectively, illustrating the different constructs used in our study. Point mutations in the DD are shown in orange.
B,ribbondiagramofdVH1(PDB3CM3)withthetwoprotomerscoloredingreenandcyan.Thedimerizationdomainofoneprotomeriscolorcodedasinpanel
A.TheselectedresiduesmutatedinAweremappedontothethree-dimensionalstructureofdVH1andareshownassticks.C,DSPcoreofVacciniaVH1(green)
is structurally superimposable to human VHZ (PDB 2IMG), considered the minimal essential core of DSPs (21) (brown).
TABLE1
Sedimentation coefficient and enzymatic activity of VH1 constructs
toward OMFP
Sedimentation
coefficient
S(20, w)
dVH1 3.24
dVH1-C110S 3.23
dVH1-2m 3.24
dVH1-5m3.29
mVH12.23
Chimeras
chVH1-CC3.17
chVH1-SS 3.18
chVH1-CS3.16
chVH1-SC3.20
VH1 constructKm
?M
Vmax
kcat/Km
M?1s?1
2.05 ? 106
87.17 ? 12
—
263.4 ? 26
351.1 ? 30
780.4 ? 152
349.5
—
619.2
563.1
26.13
1.18 ? 106
0.80 ? 106
0.017 ? 106
108.2 ? 25
—
73.63 ? 15
48.95 ? 17
495.3
—
176.3
201.9
2.29 ? 106
2.39 ? 106
4.12 ? 106
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VH1 Active Sites Are Independently Active in the Context of
the Dimeric Phosphatase—The discovery that as a monomer,
VH1 has such a dramatic reduction in catalytic activity
prompted us to investigate whether the two active sites within
the dimeric phosphatase function cooperatively. Accordingly,
loss of catalytic efficiency observed for mVH1 could be
explained by loss of positive cooperativity. This was partially
supportedbyaborder-lineHillcoefficientofn?1.2calculated
fordephosphorylationofOMFP,whichispotentiallyindicative
of positive cooperativity. To test our hypothesis, we needed to
inactivate one active site in dVH1 while leaving the second site
active. To avoid unfolding the dimer and reconstituting it with
mutatedandwtmonomersofVH1,wetookaproteinengineer-
ing approach. We generated a monocistronic chimera of VH1
that contains two VH1 genes fused by a five glycine residue
linker(Fig.4A).ThislinkerbridgestheCterminusofprotomer
FIGURE 3. Deleting residues 1–20 of VH1 results in a monomeric DSP core. A, analytical ultracentrifugation sedimentation velocity analysis. The fitted
distributionofthesedimentationcoefficientcalculatedfordVH1(2.27S)andmVH1(1.73S)correspondstoanestimatedmolecularmassof?39.6and?22.1
kDa,respectively.GiventhepredictedmolecularmassofVH1(?21kDa),thisindicatesthatinsolutionmVH1existsasamonomer.B,thermaldenaturationof
mVH1anddVH1monitoredbymeasuringchangesintheellipticityintensityat220nmasafunctionoftemperature.ApparentTmvaluesfordVH1andmVH1
are55and52 °C,respectively.C,hydrolysisofOMFP(?400?m)monitoredatanabsorbanceof477nmasafunctionoftime,inthepresenceof1?mofdVH1,
mVH1 or dephosphorylation buffer. On the right, the blow up panel shows the Michaelis-Menten saturation kinetics of mVH1 as compared with the sponta-
neous rate of OMFP hydrolysis in an aqueous environment.
VH1CatalyticActivitythroughDimerization
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A (C1) to the N terminus (N2) of protomer B, which is located
only 7 Å away in the crystal structure of dVH1 (17) (Fig. 4A).
The monocistronic chimera of dimeric VH1 (chVH1) allows
introducing point mutations in either protomer and thereby is
a useful tool to study positive cooperativity in VH1.
As a proof of concept that engineering VH1 is compatible
with the phosphatase folding, we expressed chVH1 in E. coli
and purified it under identical condition as dVH1. We did not
observed differences in expression level or solubility of chVH1
as compared with dVH1. We then introduced individual
(C3S) mutations in the active sites that destroy phosphatase
catalyticactivity.Intotal,wegeneratedfourchimerasthatcon-
taineitheridenticalactivesites(chVH1-CCandchVH1-SS),or
just individually mutated active sites (chVH1-SC and chVH1-
CS). On SDS-PAGE, all chVH1s migrated as a ?42kDa band,
exactlytwicethesizeofdVH1(Fig.4B).Theoligomericstateof
VH1chimeraswasfurtherinvestigatedbysedimentationveloc-
ity analysis (Table 1). In all cases, the sedimentation boundary
exhibits monophasic behavior (data not shown) indicative of a
single major (?99%) component in solution, migrating with a
sedimentation coefficient varying between 2.18S and 2.22S
(Table1),whichcorrespondstoamolecularmassbetween?39
and 41 kDa ? 1.5 kDa. These values agree well to a molecular
mass of ?40.5 kDa expected for dVH1. To demonstrate that
chVH1s are correctly folded, we studied the temperature-in-
duced equilibrium unfolding of purified VH1 chimeras. Varia-
tion in ellipticity at 220 nm, revealed a steep unfolding transi-
tion with an apparent temperature of melting (Tm) between
?55–68 °C (Fig. 4C). The same two-state highly cooperative
unfolding transition observed for dVH1 was also seen for all
chVH1s. For both dVH1 and chVH1, the catalytically inactive
phosphatase was significantly more stable than their catalyti-
cally active counterparts; Tmmeasured for dVH1-C110S and
chVH1-SS were 68 and 66 °C versus 55 and 56 °C of dVH1 and
chVH1-CC (Fig. 4C). The enhanced reactivity and somewhat
decreased structural stability of catalytically competent phos-
phatasesiswelldocumentedinliterature(7)andlikelyexplains
why most phosphatases have been crystallized as catalytically
inactive. Accordingly, the mixed chimeras chVH1-SC and
chVH1-CS had comparable stability (Tm?62 °C), inbetween
that of the fully inactive and active phosphatase. Thus, the chi-
meric model of VH1 reproduces faithfully the dimeric quater-
narystructureandstabilityofwtVH1,andcanbeusedtostudy
VH1 putative positive cooperativity.
We next tested if chVH1s are active toward the phosphatase
substrate OMFP. As positive control, chVH1-CC displayed
similar catalytic efficiency as wt dVH1 (kcat/Km? 2.3 ? 106
versus 2.05 ? 106) (Table 1). As expected, chVH1-SS, like
dVH1-C110S (the non-chimeric inactive dVH1) had no mea-
surable activity (Table 1). Interestingly, both chVH1-CS and
FIGURE4.EngineeringmonocistronicchimerasofdVH1thatcarrymutationsintheactivesites.A,modelofchVH1(withoneprotomeringreenandthe
otherincyan)witha5-glycinelinker(inred)bridgingtheCterminusofoneprotomertotheNterminusoftheother.B,SDS-PAGEgelshowingpurifiedwtVH1
that runs as a ?20.5 kDa species and the different monocistronic VH1 chimeras constructed in this study, which run as two fused protomers of VH1 ?41 kDa
in mass. C, stability of chimeric VH1s against thermal denaturation monitored by measuring changes in the ellipticity intensity at 220 nm as a function of
temperature. Apparent Tmvalues for chVH1-CC, dVH1, chVH1-SC, chVH1-CS, chVH1-SS, and dVH1-C110S are 55, 54, 62, 63, 65, and 68 °C, respectively.
VH1CatalyticActivitythroughDimerization
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chVH1-SCwereactivetowardOMFP,buttoadifferentextent.
Whereas chVH1-SC was twice more active than chVH1-CS
(kcat/Km?4.1?106versus2.4?106)andequallyasefficientas
chVH1-CC, it displayed nearly half of the final Vmax(Table 1).
Remarkably, all three chVH1s containing at least one catalytic
cysteinewereovertwoordersofmagnitudemoreefficientthan
mVH1. These data clearly rule out the idea of positive cooper-
ativity in VH1 catalysis, and, instead, suggests that the VH1
active sites are non equivalent and function independently.
Dephosphorylation of Activated STAT1 Requires an Intact
Dimerization Interface—All activity measurements presented
so far employed the monomeric phosphosubstrate OMFP,
which, although useful to recapitulate most monomeric sub-
strates, does not reflect the quaternary structure of dimeric
pSTAT1.Intuitively,theinteractionbetweenVH1anddimeric
pSTAT1 (?175 kDa) is likely much more complex than that
with OMFP, as pSTAT1 also binds other parts of VH1 distinct
from the active sites. To that end, we tested all VH1 constructs
designed in this study (dVH1, mVH1, VH1 chimeras,
dVH1–2m and dVH1–5m) for their ability to dephosphorylate
purifiedpSTAT1atpositionTyr-701.Usinganinvitrodephos-
phorylation assay, loss of a phosphate moiety at position 701
was determined by anti-pTyr Western blotting. In a time
course of reaction, ?80% of full-length pSTAT1 was dephos-
phorylated by dVH1 over 120 min (Fig. 5, A and B). The phos-
phataseactivitywasspecific,asthecatalyticallyinactivedVH1-
C110SshowednoappreciableSTAT1dephosphorylation,even
afterprolongedincubation(Fig.5,AandB).Indeed,thechime-
ras chVH1-CC, chVH1-SC and chVH1-CS all dephosphoryl-
ated pSTAT1 as much as 80% in 120 min, confirming compa-
rable catalytic activities to that of dVH1 (Fig. 5, A and B). In
agreementwithitspoorcatalyticactivitytowardOMFP,mVH1
had considerably reduced activity compared with dVH1 and
dephosphorylated only 20% of pSTAT1 over 120 min (Fig. 5, A
and B). Surprisingly, the constructs of VH1 with point muta-
tionsinthedimerizationdomainhadastarkreductioninphos-
phatase activity toward pSTAT1 comparable to that seen for
mVH1 (Fig. 5, A and B). This was unexpected considering that
FIGURE5.DephosphorylationassayofSTAT1atpositionTyr-701usingdifferentconstructsofVH1.A,timecourseofpSTAT1dephosphorylationinthe
presence of different constructs of recombinant purified VH1. The efficiency of dephosphorylation was monitored by anti-pTyr Western blotting. B, compar-
ativequantificationofbandintensitiesfromAusingfourindependentdephosphorylationexperimentsfor0,60,and120mintimepoints.Thegraphcoloring
has been grouped by different VH1 constructs with dVH1, mVH1, and chVH1 colored as dark gray, black, and light gray, respectively.
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both mutants are significantly more active than mVH1 toward
OMFP (Table 1) and retain a dimeric structure.
DISCUSSION
It is well documented that dimerization plays an important
role in controlling and regulating catalytic activity and sub-
strate specificity of classical PTPs (7). However, less is known
about the role of dimerization in VH1-like DSPs, which are
generally regarded as functional monomers. In this report, we
provide compelling evidence that dimerization of the Vaccinia
virus-encoded VH1 is necessary for its optimal catalytic activ-
ity. As suggested by a previous crystal structure (17), dimeric
VH1 is held together by an N-terminal dimerization domain,
that spans residues 1–27. SAXS analysis of purified VH1 con-
firmed the existence of a dimeric quaternary structure in solu-
tion. Likewise, mutations in VH1-DD reduce catalytic activity
by decreasing the enzyme affinity for phosphosubstrate. This
reduction in activity is much greater for the physiological sub-
strate, STAT1 phosphorylated at Tyr-701 than the generic col-
orimetric substrate OMFP. This is likely explained by the fact
that mutations in VH1-DD affect VH1 ability to recognize
dimeric pSTAT1, thereby reducing the phosphatase catalytic
efficiencytowardthissubstrate.Intuitively,thislossoffunction
wasnotdetectedwhenVH1catalyticactivitywasprobedwitha
small molecule compound like OMFP.
Deletion of VH1 swapped helix spanning residue 1–20,
which builds much of the dimerization interface, results in a
monomeric phosphatase that is ?120-fold less active than
dVH1 and, at best, dephosphorylates 20% of pSTAT1 in a time
course of 120 min. The dramatic difference in catalytic activity
between monomeric and dimeric VH1 cannot be ascribed
uniquely to a loss of structural stability of mVH1 as compared
with dVH1. The difference in (apparent) Tmbetween the two
proteins is only 3 °C (?52 °C versus ?55 °C), although the two
phosphatases unfold in a remarkably different way. Thus, the
minimal DSP core (aa 28–171) of VH1 is only minimally active
in the absence of its DD. This is in stark contrast to other VH1-
likephosphataseslikeVHZ(alsoknownasDUSP25),forwhich
the minimal ?16 kDa DSP core carries all structural and func-
tional determinants sufficient for catalytic activity (33). As a
corollary, the minimal sets of secondary structural elements
conserved in all knownVH1-like DSPs (5 ?-helices and 5
?-strands) as well as the presence of a catalytic triad (Cys/Arg/
Asp) are not sufficient requirements for a catalytically active
DSPcore.mVH1containsafoldedandstableDSPcore,butitis
minimally active. Dimerization via the N-terminal swapped
dimerization domain appears essential for VH1 optimal cata-
lytic activity. Moreover, as demonstrated by our chimeric
model of dVH1, a “single active” active site within the same
VH1dimerissufficientforcatalyticactivitytowardbothOMFP
and pSTAT1. This rules out the idea of positive cooperativity
between the two VH1 active sites and further emphasizes the
importance of an intact dimerization interface. Accordingly,
pointmutationsattheVH1dimerizationinterfacethatalterthe
interface compactness without disrupting dimerization have a
destructiveeffectoncatalysis.Itisimportanttonotethatwhile
the point mutants dVH1–2m and dVH1–5m demonstrate
much higher efficiencies than mVH1 when tested for OMFP
dephosphorylation,themutants(especiallydVH1–5m)arejust
asineffectiveasmVH1atdephosphorylatingpSTAT1(Fig.5B).
Thus, even if these mutants contain wt catalytic sites, simple
destabilization of the dimerization interface results in a dra-
matic reduction of pSTAT1 dephosphorylation. Having estab-
lishedthatVH1-DDdoesnotmediatecross-talkbetweenactive
sites through positive cooperativity, the sensitivity of this
dimerizationinterfacetomutationslikelyreducesthephospha-
tase’s ability to recognize and bind pSTAT1. Furthermore, the
observationthatonlyoneactivesitewithinVH1dimericstruc-
ture is sufficient for catalysis indirectly suggests that this phos-
phatase functions as a distributive enzyme, which associates to
the substrate, dephosphorylates at least one Tyr-701, followed
by dissociation and a new round of catalysis. Dimerization
likely affects the first step of this process, the recognition of
pSTAT1, as well as it enhances VH1 intrinsic phosphatase
activity, as demonstrated in this report.
Analogies with Human Laforin—Liu et al. (34) reported that
dimerization of the VH1-like DSP laforin is essential for its
catalyticactivity.Laforinisa?331aminoacidphosphatasethat
contain an N-terminal carbohydrate binding module (CBM)
fused by to a DSP domain, 21% similar to that of VH1 (35) (Fig.
6).MutationsinthelaforingenehavebeenassociatedtoLafora
disease (LD), a fatal neurodegenerative disorder characterized
by the accumulation in the brain of polyglucosan aggregates,
named Lafora Bodies (35). The observation that laforin
dimerization is essential for its catalytic activity (34) is impor-
tant to explain why mutations outside the laforin DSP core
reducephosphataseactivity,therebyleadingtolossoffunction.
An interesting parallelism that emerges from our and Liu
work (34) is that both laforin and VH1 share a fundamentally
similarstructuraltopologyandrelyondimerizationtobefunc-
tional (Fig. 6). This has at least three functional implications,
pertinent to the biology of DSPs. First, in both phosphatases,
the minimal DSP core is in the C terminus of the protein, while
an N-terminal domain serves as a dimerization domain.
Although VH1-DD is only ?27 residues, in laforin this domain
FIGURE 6. Schematic cartoon illustrating the basic domain organization
of dimeric laforin and VH1. In both cases the two phosphatase protomers
contain an N-terminal dimerization domain (DD and CBM in VH1 and laforin,
respectively) fused to a C-terminal DSP core. In the diagram, the N-terminal
dimerizationdomainisswappedbothinVH1andlaforin,althoughithasnot
been demonstrated for the latter. Active sites are represented by orange
circles.
VH1CatalyticActivitythroughDimerization
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is larger (?90 residues) and also binds carbohydrates (35).
Deleting the laforin N-terminal CBM abolishes dimerization
(34), suggesting that this domain, and not the DSP, is responsi-
ble for dimerization. Equally in VH1, removing the N-terminal
swapped helix (aa 1–20) renders the phosphatase monomeric.
Second, in both laforin and VH1, the DSP core is not sufficient
per se for efficient catalysis, yet its activity is greatly stimulated
by a dimeric quaternary structure, mediated by the N-terminal
DD.Itcouldbespeculatedthat,likeinVH1,laforinN-terminal
CBMs are also swapped between two identical protomers (Fig.
6). Although not necessary for dimerization, domain swapping
is a potentially convenient way to enhance enzyme specificity
for substrate (36) and facilitate phosphosubstrate presentation
to the active site. Third, and finally, both laforin and VH1 sub-
strates present a modular and symmetric structure. Laforin
dephosphorylates glycogen (10), which is formed by repetitive
units of glucose, whereas VH1 dephosphorylates activated
STAT1 phosphorylated at Tyr-701 (17, 20). A dimer of VH1
exposestwoactivesitesspaced?39Åawayfromeachotheron
the surface of the phosphatase (Fig. 2B), which is also the dis-
tance between two pTyr-701 in a dimer of STAT1 (37). We
propose that the three-dimensional complementarity between
a dimeric DSP and its dimeric substrate is a potential determi-
nant for specificity.
In conclusion, dimerization of VH1-like DSPs emerges as a
distinct and vital structural requirement for certain DSPs to
adoptanactivequaternarystructure.Futurestudieswillhaveto
determine if dimerization plays a role in other DSPs that are
commonly assumed to be monomeric. Furthermore, it will be
interestingtodetermineifdimerizationcanmodulatesubstrate
specificity,inadditiontobeessentialforoptimalcatalyticactiv-
ity, as demonstrated in this report.
Acknowledgments—We thank members of the Cingolani Laboratory
for critical reading of the manuscript. We are thankful to Marc
Allaire and the staff at NSLS beamline X9 for beamtime and help in
data collection.
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