Biochemical properties of the human guanylate binding
protein 5 and a tumor-specific truncated splice variant
Mark Wehner and Christian Herrmann
Ruhr-Universita
¨
t Bochum, Physikalische Chemie I – AG Proteininteraktionen, Universtita
¨
tsstraße 150, Bochum, Germany
Introduction
A large variety of cellular processes, including signal
transduction, regulation of transcription and membrane
deformation, are regulated by GTP-binding proteins
[1,2]. Usually these proteins act as ‘molecular switches’
that strongly interact with their effector proteins in the
GTP-bound state (‘on’ state) but only very weakly in
their GDP-bound state (‘off’ state) [3]. By hydrolysis of
the GTP to GDP and P
i
, these proteins are able to
‘switch off’ intrinsically, or GTP cleavage may be
promoted by interaction with a GTPase-activating
protein. Small regulatory GTP-binding proteins from
the Ras superfamily usually only exchange the bound
nucleotide very slowly, but the exchange rate is dramati-
cally increased in the presence of guanine nucleotide
exchange factors, and can be regulated. GTPases from
the dynamin superfamily (reviewed in [4]), on the other
Keywords
biophysics; dynamin; GTPase; guanylate
binding protein; interferons
Correspondence

ing changes in the oligomerization state. We observed only a minor influ-
ence of the C-terminal truncation on hydrolysis, nucleotide binding and
oligomerization of hGBP5. Based on these similarities we speculate that the
missing C-terminal part, which also carries the geranylgeranylation motif, is
the reason for the dysregulation of hGBP5¢s function in lymphoma cells.
Structured digital abstract
l
MINT-7555035, MINT-7555053: hGBP5 a ⁄ b (uniprotkb:Q96PP8) and hGBP5 a ⁄ b (uni-
protkb:
Q96PP8) bind (MI:0407)bymolecular sieving (MI:0071)
l
MINT-7555028, MINT-7555044: hGBP5ta (uniprotkb:Q86TM5) and hGBP5ta (uni-
protkb:
Q86TM5) bind (MI:0407)bymolecular sieving (MI:0071)
Abbreviations
GppNHp, guanosine 5¢-(b,c-imino)-triphosphate; GTPcS guanosine, 5¢-O-(c-thio)triphosphate; hGBP, human guanylate binding protein; ITC,
isothermal titration calorimetry; mant, N-methylanthraniloyl.
FEBS Journal 277 (2010) 1597–1605 ª 2010 The Authors Journal compilation ª 2010 FEBS 1597
hand, interact dynamically with the bound nucleotide,
and usually bind nucleotides in the micromolar range.
In addition to these striking differences in nucleotide
binding, these large GTPases are catalytically very
active, show self-activating GTP hydrolysis, and are
functional as oligomers rather than monomers.
Human guanylate binding protein 5 (hGBP5) belongs
to the family of interferon-c-induced p65 GTPases,
which has seven members in the human genome [5].
This family of guanylate binding proteins was origi-
nally identified by its ability to bind to immobilized
guanine nucleotides with similar affinities for GTP,

to exist as more than one splice variant, hGBP5 does
exist as three splice variants, which form two different
proteins [16]. These two proteins differ with respect to
the presence or absence of the C-terminal 97 amino
acids including the C-terminal geranylgeranylation
motif. If a fold similar to that of hGBP1 is assumed,
this deletion corresponds to deletion of extended helices
12 and 13. In healthy cells, only the a ⁄ b splice variant
is expressed, but the truncated splice variant has been
detected in all melanoma and most of lymphoma cell
lines tested.
In this study, we focus on the biochemical properties
of both splice variants of hGBP5, hGBP5a ⁄ b (amino
acids 1-586) and the C-terminally truncated hGBP5ta
(amino acids 1-489), using isothermal titration calorim-
etry (ITC), concentration-dependent GTPase assays,
fluorescence titrations and analytical gel filtration.
Results and Discussion
Hydrolytic activity of hGBP5a/b and hGBP5ta
In light of previous observations for hGBP1 and other
large GTPases, we analysed the enzymatic activity of
hGBP5a ⁄ b and hGBP5ta in a concentration-dependent
manner. Various concentrations of purified hGBP5a ⁄ b
and hGBP5ta were incubated with 350 lm of GTP,
and aliquots were taken after various durations and
analysed by C18 reverse-phase HPLC. Initial rates of
GTP hydrolysis were normalized to the protein con-
centration (specific activity) and plotted against the
protein concentration (Fig. 1).
We observed weak concentration-dependent self-acti-

(see Fig. S1). As described previously [18], certain posi-
tions in the hGBP1 large GTPase domain are crucial
for the formation of GMP by hGBP1. Surprisingly,
these residues (namely R48, H74, K76, E99, K106 and
D112 in the primary sequence of hGBP1) are con-
served in the primary structure of the GTPase domain
of hGBP5, so the formation of GMP must be impaired
in an as yet unknown manner. Sequence alignments of
the large GTP-binding domains of hGBP1, hGBP2
and hGBP5 showed many substitutions specific to
hGBP5, so we cannot conclude at present which resi-
dues are responsible for the lack of GMP formation
(see Fig. S2). The similar hydrolytic activities of the
a ⁄ b and C-terminally truncated forms of hGBP5 are in
contrast to that of the analogous deletion mutant of
hGBP1 (S. Kunzelmann, Ruhr-University Bochum and
C. Herrmann, unpublished results), which resulted in a
2.5-fold increase in specific GTPase activity and
increased formation of GMP during hydrolysis.
Thermodynamics and stoichiometry of nucleotide
binding
We used ITC to investigate the thermodynamics of
nucleotide binding. Using this method, it is possible
to determine the stoichiometry, enthalpy change,
dissociation constant and thereby the change in
entropy in a single experiment. No label is required
in these experiments as the heat change of the
reaction is measured.
Using ITC, we found a 1 : 1 stoichiometry of nucle-
otide to protein with a deviation of less than 10%. We

)1
).
The TÆDS values for GDP are less negative (> )10 kca-
lÆmol
)1
) for both splice variants. We attribute these pro-
nounced entropic compensations to a loss of
conformational freedom upon nucleotide binding, and,
especially when using the GTP analogs, to the forma-
tion of higher-order oligomers. In the case of hGBP1,
the entropy changes for nucleotide binding are generally
positive but less than 5 kcalÆmol
)1
. Despite the fact that
both hGBP5 and hGBP1 form oligomers in a nucleo-
tide-dependent manner, the entropic contributions of
nucleotide binding and the coupled oligomerization
show opposing signs. In the case of hGBP1, release of
water from the nucleotide binding pocket and the pro-
tein surface may counterbalance the changes in confor-
mational flexibility imposed by nucleotide binding,
while in the case of hGBP5, conformational restrictions
after nucleotide binding and oligomer formation appear
to result in strong entropic penalties.
In contrast to the findings for hGBP1 [8], we did
not observe any signals in ITC experiments when using
GMP. Neither the a ⁄ b form nor the truncated splice
variant exhibit any GMP binding in the micromolar
range (see Fig. 2 for a representative experiment). The
missing signals in our ITC experiments are not due to

observed, as described previously for hGBP1 [8]. The
observed approximately 3.5-fold increase in fluores-
cence is indicative of a non-solvent-accessible binding
pocket, as found in the crystal structure of hGBP1
[20,21]. Analysis of the fluorescence intensities using a
quadratic binding equation yielded the dissociation
constants summarized in Table 2. The splice variants
hGBP5a ⁄ b and hGBP5ta did not show any significant
differences in mant-nucleotide binding. The observed
dissociation constant for mant-GTPcS (11 lm)is
higher than that for mant-GDP (3 lm), which is the
converse of the tendency observed with unlabeled
nucleotides in ITC experiments (see Fig. 3). To further
confirm the lack of GMP binding by hGBP5 splice
variants as observed in the ITC experiments, we per-
formed fluorescence titrations using mant-GMP. Simi-
lar to the ITC experiments with GMP, we only
observed a small effect, i.e. a marginal increase in
mant-GMP fluorescence in the presence of 50 lm
hGBP5 (data not shown). Furthermore, we attempted
to displace bound mant-GDP using a high excess
(3000-fold) of GMP, but only observed a decrease in
fluorescence of less than 10%, indicating a much
higher dissociation constant for GMP than for mant-
GDP. In contrast, displacement of mant-GDP from
hGBP5 was efficient at low concentrations of compet-
ing GDP, GppNHp and GTPcS (data not shown).
When investigating mant-GppNHp binding to
hGBP5a ⁄ b or hGBP5ta, we found very low reaction
rate constants that were at least 100-fold smaller than

respectively. Relative fluorescence values are plotted against the
concentration of added protein.
Biophysical properties of hGBP5a ⁄ b and hGBP5ta M. Wehner and C. Herrmann
1600 FEBS Journal 277 (2010) 1597–1605 ª 2010 The Authors Journal compilation ª 2010 FEBS
To investigate the dynamics of nucleotide binding,
constant concentrations of the nucleotides were mixed
with increasing amounts of hGBP5a ⁄ b or hGBP5ta
(see Fig. 4 for representative data). In the case of
mant-GTP, increasing amounts of mant-GTP were
mixed with a small concentration of hGBP5a ⁄ b
or hGBP5ta to avoid fluorescence changes due to
nucleotide hydrolysis.
The association experiments for all nucleotides mea-
sured only show a single rate constant. In contrast, the
displacement experiments using mant-GTPcS revealed
two distinct rate constants for dissociation, with the
relative amplitudes of 60% for the faster process and
40% for the slower process, which is indicative of a
difference in binding dynamics of the 2¢⁄3¢-OH mant-
labeled GTPcS as the two isomers occur in approxi-
mately this ratio. We did not find any evidence for
two dissociation rates for mant-GDP (see Fig. S3).
Using hGBP5, we found rate constants that were
approximately 100-fold lower for mant-GTP and
approximately 30-fold lower for mant-GDP than those
reported for hGBP1 [9]. The association rate constants
(k
on
) for mant-GDP (0.046 lm
)1

)1
Æs
)1
, respectively. The dissociation rate con-
stant obtained from the intercept of the plot in Fig. 4,
parts B and C (k
intercept
off
) and the directly measured dis-
sociation rate constants from displacement experiments
Fig. 4. Nucleotide dynamics determined using stopped flow mea-
surements. (A) Representative fluorescence traces of mant-GDP
binding to hGBP5ta. The hGBP5 concentrations increase from
2.5 l
M to 25 lM from the bottom up. (B) The observed rates of
mant-GDP (filled circles) and mant-GTPcS (open circles) association
are plotted against the protein concentration. (C) Observed associa-
tion rates of mant-GTP with hGBP5a ⁄ b (open circles) or hGBP5ta
(filled circles).
Table 3. Nucleotide binding dynamics of hGBP5a ⁄ b and hGBP5ta.
The dissociation constants were calculated using the relationship
K
d
= k
off
diss
⁄ k
on
, except for mant-GTP, for which k
off

1.9 ± 0.3
mant-GTP 0.014 ± 0.001 0.62 ± 0.01 – 44 ± 4*
hGBP5ta
mant-GDP 0.066 ± 0.004 0.13 ± 0.01 0.23 ± 0.01 3.5 ± 0.3
mant-GTPcS 0.072 ± 0.006 1.4 ± 0.1 1.15 ± 0.05
a
16 ± 2
0.20 ± 0.03
b
2.8 ± 0.7
mant-GTP 0.025 ± 0.005 0.72 ± 0.07 – 30 ± 10
*
a
Corresponding relative amplitude = 0.6.
b
Corresponding relative
amplitude = 0.4.
M. Wehner and C. Herrmann Biophysical properties of hGBP5a ⁄ b and hGBP5ta
FEBS Journal 277 (2010) 1597–1605 ª 2010 The Authors Journal compilation ª 2010 FEBS 1601
(k
diss
off
) in Fig. S3 are similar, and show a five- to
10-fold lower rate for dissociation of mant-GDP
(0.129 ⁄ 0.23 s
)1
) from hGBP5ta than for mant-GTPcS
(1.15 s
)1
), and the value for mant-GTP is between

. Because of the unexpected
observation of a monomeric protein species with both
GDP and GDP ÆAlF
x
, we tested AlF
x
binding to the
protein by adding 300 lm AlCl
3
and 10 mm NaF to a
solution containing hGBP5a ⁄ b or hGBP5ta and GTP,
and analysed GTPase activity. We observed a reduc-
tion of GTPase activity of approximately 40%. This
small inhibitory effect suggests weak binding of AlF
x
to the protein. In the GTP-bound (as well as the nucle-
otide-free) state, a M
app
of approximately 120 kDa is
observed, which probably corresponds to a dimer.
When bound to GppNHp, the M
app
of hGBP5ta is
increased to approximately 200 kDa, which corre-
sponds to a tetramer, while the presence of GTPcS
leads to an oligomer size intermediate between those
with GTP and GppNHp (see Fig. 5A). The putative
transition state hGBP5ÆGDPÆAlF
x
resembles the prod-

Fig. 5. Size-exclusion chromatography of the splice variants
hGBP5ta (A) and hGBP5a ⁄ b (B), respectively, with various nucleo-
tides [red, GDP; green, GDPÆAlF
x
; magenta, GTP; blue, GppNHp;
cyan, GTPcS] and in the nucleotide-free state (black). The absor-
bance at 280 nm was plotted against the elution volume (V
e
)
normalized to the exclusion volume (V
0
).
Biophysical properties of hGBP5a ⁄ b and hGBP5ta M. Wehner and C. Herrmann
1602 FEBS Journal 277 (2010) 1597–1605 ª 2010 The Authors Journal compilation ª 2010 FEBS
dimer, resulting in monomeric product complex in the
case of hGBP5taÆGDP. In contrast, the hGBP5a ⁄ b
splice variant remains dimeric. This difference may be
caused by the additional amino acids at the C-terminus
of hGBP5a ⁄ b.
Concluding remarks
We observed biochemical properties for hGBP5
that are strongly different from those of the well-
characterized isoform hGBP1. The GTPase activity is
much lower, and, in particular, the concentration-
dependent activation is not as pronounced. In contrast
to hGBP1, we did not observe any GMP binding by
hGBP5, and the enzymatic hydrolysis of GTP leads to
GDP rather than GMP. The nucleotide affinities of
hGBP5 are grossly similar to those of hGBP1, but
there are differences in thermodynamic and kinetic

2
,2mm dithiothreitol).
Pure protein fractions were concentrated to approximately
1mm, frozen in liquid nitrogen, and stored at )80 °C.
Concentrations of the purified proteins were measured
using UV absorbance at 280 nm (e = 45 380 and 38 880
(mÆcm)
)1
for hGBP5a ⁄ b and hGBP5ta, respectively).
Hydrolysis assays
Hydrolysis measurements were performed as described
previously [19] using 350 lm GTP (Sigma-Aldrich,
Munich, Germany) and increasing concentrations of
hGBP5a ⁄ b or hGBP5ta in buffer C containing 50 lm BSA
(Sigma-Aldrich) for protein stabilization at 25 °C. Aliquots
were taken after defined incubation periods, injected onto a
Chromolith RP18e HPLC column (Merck), and elution
was followed by determination of absorption at 254 nm
using an MD5100plus diode array detector (Jasco, Gross-
Umstadt, Germany). The running buffer was composed of
100 mm potassium phosphate, 10 mm tetrabutylammonium
bromide, 0.2 mm sodium azide and 1.25% acetonitrile at
pH 6.5. Elution times were measured using GMP, GDP
and GTP (all purchased from Sigma-Aldrich) as calibration
standards. Data were fitted using the Hill-like model shown
in Eqn (1) where S
min
and S
max
represent minimum

an ITC-Origin calorimeter (Microcal ⁄ GE Healthcare).
Size-exclusion chromatography
Analytical gel filtration experiments were performed using a
Superdex 200 10 ⁄ 300 column (GE Healthcare). The elution
buffer (50 mm Tris pH 7.9, 5 mm MgCl
2
, 150 mm NaCl)
contained 200 lm of the nucleotide, and 300 lm AlCl
3
and
10 mm NaF were added in the case of GDPÆAlF
x
. Protein
(20 lm) was preincubated in the elution buffer for 30 min
on ice before being injected onto the gel filtration column.
Size calibration was carried out using standard proteins
with masses in the range of 29–669 kDa (the corresponding
elution volumes are indicated on the graphs by arrows).
M. Wehner and C. Herrmann Biophysical properties of hGBP5a ⁄ b and hGBP5ta
FEBS Journal 277 (2010) 1597–1605 ª 2010 The Authors Journal compilation ª 2010 FEBS 1603
Elution was followed by monitoring the absorbance at
280 nm using an A
¨
kta Purifier system (GE Healthcare).
Fluorescence titration
Fluorescence titrations were performed at 25 °C using an
SFM25 fluorospectrometer (Kontron, Zurich, Switzerland)
and mant-labeled nucleotides (Jena Biosciences). The excita-
tion and emission wavelengths were 366 and 435 nm,
respectively. mant-labeled nucleotide (0.5 lm) was titrated

on
), and the intercept rep-
resents the dissociation rate (k
off
). In the case of displace-
ment experiments, 10 lm protein was preincubated with
0.5 lm of mant-nucleotide. The mant-nucleotide was dis-
placed by mixing with a 1000-fold excess of unlabeled
GDP, and the resulting rate constant corresponds to k
diss
.
Fluorescence traces were fitted using a single rate constant
(mant-GDP) or two rate constants (mant-GTPcS). Corre-
sponding dissociation constants are calculated from the
relationship K
d
= k
off
⁄ k
on
.
Acknowledgements
We thank Professor Dr Michael Stu
¨
rzl for providing
the cDNA for hGBP5, the Deutsche Forschungsgeme-
inschaft for financial support, and the Ruhr-University
Research School for a full scholarship to M.W.
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Supporting information
The following supplementary material is available:
Fig. S1. GTP hydrolysis at 37 °C.
Fig. S2. Sequence alignment of the hGBP1 ⁄ 2 ⁄ 5LG
domains.
Fig. S3. Displacement experiments.
This supplementary material can be found in the
online version of this article.
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