Báo cáo khoa học: Biochemical properties of the human guanylate binding protein 5 and a tumor-specific truncated splice variant Mark Wehner and Christian Herrmann doc

In contrast to the small regulatory GTP-binding proteins of the Ras superfamily, hGBP1 is character-ized by highly dynamic nucleotide binding, low affi-nities for all three guanine nucleo

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 Pi, 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

C Herrmann, Ruhr-Universita¨t Bochum,

Physikalische Chemie I – AG

Proteininteraktionen, Universita¨tsstraße 150,

44800 Bochum, Germany

Fax: +49 2343214785

Tel: +49 2343224173

E-mail: chr.herrmann@rub.de

(Received 9 October 2009, revised 15

January 2010, accepted 19 January

2010)

doi:10.1111/j.1742-4658.2010.07586.x

The human guanylate binding protein 5 (hGBP5) belongs to the family of interferon-c-inducible large GTPases, which are well known for their high induction by pro-inflammatory cytokines The cellular role of this protein family is unclear at this point, but there are indications for antiviral and antibacterial activity of hGBP1 hGBP5 exists in three splice variants, form-ing two different proteins, of which the tumor-specific one is C-terminally truncated by 97 amino acids, and therefore lacks the CaaX motif for gera-nylgeranylation Here we present biochemical data on the splice variants of hGBP5 We show that, unlike hGBP1, hGBP5a⁄ b and hGBP5ta do not bind GMP or produce any GMP during hydrolysis despite the fact the resi-dues involved in GMP production from hGBP1 are conserved in hGBP5 Hydrolysis of GTP is concentration-dependent and shows weak self-activa-tion Thermodynamic studies showed strongly negative entropic changes during nucleotide binding, which reflect structural ordering in the protein during nucleotide binding These structural changes were also observed dur-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) by molecular sieving (MI:0071)

l MINT-7555028, MINT-7555044: hGBP5ta (uniprotkb:Q86TM5) and hGBP5ta (uni-protkb:Q86TM5) bind (MI:0407) by molecular 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.

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,

GDP and GMP [6,7] Within this family, the best

characterized member, human guanylate binding

pro-tein 1 (hGBP1), has been shown to exhibit a unique

hydrolytic activity – cleavage of not only the

c-phos-phate but also the b-phosc-phos-phate in a successive step

[8,9] In contrast to the small regulatory GTP-binding

proteins of the Ras superfamily, hGBP1 is

character-ized by highly dynamic nucleotide binding, low

affi-nities for all three guanine nucleotides, and high,

self-activated GTPase activity, which is promoted by

olig-omerization These features are generally conserved in

high-molecular-weight GTPases such as dynamin and

the antiviral protein, Mx Because of similarities in

the molecular architecture of GBPs and members of

the dynamin family, GBPs are classified as a part of

the dynamin superfamily Although their biochemical

characteristics are well understood, there is only an

incomplete picture of the cellular functions of

hGBP1 Previous studies showed an antiviral effect

against specific viruses [10,11], and an anti-Chlamydia

effect [12], inhibition of endothelial cell proliferation

[13] and their subcellular localization [14] have also

been investigated recently Similar to the recent report

of an anti-Chlamydia effect of hGBP1, there are

indications of murine GBP5 co-localization with

mem-brane ruffles formed by invading Salmonella enterica,

and a positive regulation of pyroptosis to defend

against infection by S enterica and possibly other

bacterial pathogens [15] Although no other proteins

from the guanylate-binding protein family are known

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-vation (approximately two-fold) from a basal specific activity at low protein concentrations to maximum turn-overs at 25C of 0.054 and 0.077 s)1 for hGBP5a⁄ b and hGBP5ta, respectively Increasing the temperature from 25C to 37 C resulted in an approximately three-fold increase in specific activity (see Fig S1) Both splice variants exhibited positive cooperativity in specific activity, with Hill coefficients of 2.4 and 1.9 and dissoci-ation constants (KdHill) of 4 and 2 lm for hGBP5a⁄ b and hGBP5ta, respectively The proteins are very similar

in terms of their enzymatic activity, so the activity is not affected by deletion of the C-terminal part In contrast

Fig 1 Initial rates of GTP hydrolysis were normalized to the pro-tein concentration (specific activity), and plotted against the propro-tein concentration Both hGBP5a ⁄ b (circles) and hGBP5ta (squares) show an approximately two-fold self-activation of GTP hydrolysis.

to results obtained for hGBP1 and hGBP2 [17], we

observed absolutely no formation of GMP at any

pro-tein concentration or time, indicating a different

mech-anism of hydrolysis compared to hGBP1 To confirm

that hGBP5 does not require higher temperatures to

form GMP, we performed additional hydrolysis

mea-surements at 37C, but did not detect GMP formation

(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

found comparable dissociation constants for GDP and

GppNHp [guanosine 5¢-(b,c-imino)-triphosphate], with

Kd values of 11 and 5 lm for hGBP5ta and 7.2 and

2.6 lm for hGBP5a⁄ b, respectively For the

non-hydro-lysable analog GTPcS [guanosine

5¢-O-(c-thio)triphos-phate], the dissociation constant is slightly higher (see

Table 1) In all experiments, nucleotide binding yielded

large exothermic peaks, with corresponding DH values

ranging from )14.7 kcalÆmol)1 for GDP binding to

hGBP5a⁄ b and)38.1 kcalÆmol)1for binding of GTPcS

to hGBP5ta In light of the thermodynamic data and

the oligomerization behavior, we attribute the rather

pronounced negative changes in enthalpy not simply

to nucleotide binding, but to a combination of several

exothermic effects occurring at the same time, most likely nucleotide binding and coupled oligomerization All nucleotide binding experiments showed a negative change in DS, which is compensated for by the large and negative DH value This negative change of entropy

is most pronounced for hGBP5a⁄ b using the GTP ana-log GppNHp (TÆDS =)23.5 kcalÆmol)1) and for hGBP5ta using GTPcS (TÆDS =)31.9 kcalÆmol)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

a very low DH at 25C, but are due to the absence of GMP binding, as confirmed by competitive fluor-escence measurements

Binding and dynamics of fluorescent nucleotide analogs

To investigate the binding of fluorescent nucleotide analogs, we used fluorescence titrations as described

Table 1 Thermodynamic parameters of nucleotide binding by hGBP5a ⁄ b and hGBP5ta at 25 C as measured by ITC.

Nucleotide K d (l M ) DH (kcalÆmol)1) TÆDS (kcalÆmol)1) hGBP5a ⁄ b

GppNHp 2.6 ± 0.3 )31.3 ± 0.4 )23.5 ± 0.6

hGBP5ta

GppNHp 5 ± 0.2 )25.3 ± 0.3 )18.2 ± 0.3

previously for hGBP1 [8,19] When titrating increasing

amounts of hGBP5a⁄ b or hGBP5ta with a solution

of N-methylanthraniloyl-GDP (GDP) or

mant-GTPcS, an increase in fluorescence intensity was

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 for all other nucleotides tested (data not shown) Because of the slow reaction of mant-GppNHp and hGBP5, fluorescence measurements were not feasible due to long-term protein instability Similar to these fluorescence experiments, a rather slow return to base-line in the ITC experiments was observed, but, without the mant label, GppNHp binding is fast enough to allow equilibration after each injection

Fig 2 Representative isothermal titration calorimetry runs using

hGBP5ta at 25 C hGBP5ta (150 l M ) was titrated with 2.25 m M of

nucleotide (GMP, open circles; GDP, filled squares; GppNHp, open

squares) The raw experimental data and the processed data

(including the fitted curves using a one-site binding model) are

shown in the upper and lower panels, respectively.

Table 2 Dissociation constants of mant-labeled nucleotides as

measured by fluorescence titrations.

Nucleotide K dhGBP5a⁄ b(l M ) K dhGBP5ta(l M )

Fig 3 Fluorescence titration data showing similar dissociation constants for binding of mant-GDP (hGBP5a ⁄ b, filled circles; hGBP5ta, open circles) and for binding of mant-GTPcS (hGBP5a ⁄ b, filled squares; hGBP5ta, open squares) to hGBP5a ⁄ b or hGBP5ta, respectively Relative fluorescence values are plotted against the concentration of added protein.

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 (kon) for mant-GDP (0.046 lm)1Æs)1), mant-GTPcS (0.099 lm)1Æs)1) and mant-GTP (0.014 lm)1Æs)1) were quite similar In contrast, we found a five-fold faster dissociation of mant-GTPcS (1.11 s)1) compared to mant-GDP (0.23 s)1) and mant-GTP (0.62 s)1) The three-fold lower dissociation rate (koff) for mant-GTPcS was comparable to that for mant-GDP, but is not due to partial hydrolysis of the nucleotide, as veri-fied by HPLC analysis (Table 3) We found association rate constants for hGBP5ta with GDP, mant-GTPcS and mant-GTP of 0.066, 0.072 and 0.025 lm)1Æs)1, respectively The dissociation rate con-stant obtained from the intercept of the plot in Fig 4, parts B and C (kinterceptoff ) 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 l M 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

Kd= koffdiss ⁄ k on , except for mant-GTP, for which koffintercept was used.

Nucleotide k on (l M )1Æs)1)

koffintercept

(s)1) k offdiss(s)1) K d (l M ) hGBP5a ⁄ b

mant-GDP 0.046 ± 0.003 0.28 ± 0.04 0.23 ± 0.02 5.0 ± 0.8 mant-GTPcS 0.099 ± 0.003 2.08 ± 0.04 1.11 ± 0.01a 11.2 ± 0.5

0.19 ± 0.02 b 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 ± 0.03b 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.

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

those two values (0.7 s)1) Similarly, the slower

dissoci-ation rate constant for mant-GTPcS was comparable

to that for mant-GDP (see Table 3)

Oligomerization of hGBP5a/b and hGBP5ta

Large GTPases, especially of the dynamin superfamily,

are well known for their ability to oligomerize and the

resulting stimulation of their hydrolytic activity In

particular, hGBP1 is known to form homodimers after

GppNHp binding and larger oligomers with the

transi-tion state analog GDP and aluminum fluoride (AlFx)

[18] We used analytical size-exclusion chromatography

to characterize the nucleotide-dependent

oligomeriza-tion state of the protein Protein (20 lm) was

preincu-bated with a 10-fold excess of the nucleotide for

30 min on ice prior to injection onto a Superdex 200

column equilibrated with buffer containing 200 lm of

nucleotide

The C-terminally truncated splice variant hGBP5ta

(theoretical molecular mass 55 kDa) shows an

appar-ent molecular mass (Mapp) in the GDP-bound form of

approximately 70 kDa, most likely due to an elongated

shape of the protein The same behavior is observed in

the presence of GDPÆAlFx Because of the unexpected

observation of a monomeric protein species with both

GDP and GDPÆAlFx, we tested AlFx binding to the

protein by adding 300 lm AlCl3and 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 AlFx

to the protein In the GTP-bound (as well as the

nucle-otide-free) state, a Mapp of approximately 120 kDa is

observed, which probably corresponds to a dimer

When bound to GppNHp, the Mapp 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 hGBP5ÆGDPÆAlFx resembles the

prod-uct state more closely than a GTP-bound form does

Similar to the monomeric species for the GDP-bound

state of hGBP5ta, a recent study found that dynamin

oligomers are repeatedly dissociated while GTP

hydro-lysis occurs [22]

In contrast to hGBP5ta, the hGBP5a⁄ b splice

vari-ant does not show a monomer species in the presence

of any nucleotide When bound to GDP or GDPÆAlFx,

a dimeric form is observed (taking elongated shape into account) The size of the protein complex is only slightly increased when nucleotide-free or in the presence of GTP As with hGBP5ta, the complex is increased to the size of a tetramer after binding of GppNHp, and binding of GTPcS again shows an intermediate behavior (Fig 5B) The elution profiles of both hGBP5ta and hGBP5a⁄ b in the presence of vari-ous nucleotides show a small fraction of protein com-plexes eluting at higher apparent masses This might

be an indication that equilibria between various oligo-mer complexes are established that are controlled by the bound nucleotide In contrast to hGBP1, our observations suggest that, in the course of hGBP5 cat-alyzed GTP hydrolysis, a nucleotide-free hGBP5 dimer binds GTP, leading to a larger complex (represented

by GppNHp⁄ GTPcS) GTP cleavage leads to dissocia-tion of the protein complex shown by the apparent

A

B

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 (Ve) normalized to the exclusion volume (V 0 ).

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

details The entropy changes are more negative in the

case of hGBP5, possibly reflecting larger structural

changes accompanying nucleotide binding The rate

constants for nucleotide association and dissociation

are both much smaller for hGBP5 compared to

hGBP1, reflecting a less dynamic behavior but leading

to similar binding strength Intriguingly, all

biochemi-cal parameters addressed in this work and summarized

above are similar for hGBP5a⁄ b and the splice variant

truncated at the C-terminus At this point, we can only

speculate about the origin of the difference in cells,

which is not based on different enzymatic activities

or different characteristics with respect to nucleotide

binding and subsequent formation of oligomers

Rather we assume that lack of the prenylation site in

hGBP5ta is responsible for some of the alteration of

the biological function

Experimental procedures

Expression and purification

hGBP5a⁄ b (GenBank accession number AAN39036.1) and

hGBP5ta (GenBank accession number AAO40731.1) were

subcloned into pQE80L expression vectors (Qiagen, Hilden,

Germany), transformed into Escherichia coli Rosetta 2

(DE3) pLysS (Merck, Darmstadt, Germany), grown in TB

medium to an attenuance at 600 nm of 0.6, and expression

induced by adding 100 lm isopropyl thio-b-d-galactoside

(AppliChem, Darmstadt, Germany) The final step of

purification, which has been described previously for

hGBP1 [8], was size-exclusion chromatography using buffer

C (50 mm Tris pH 7.9, 5 mm MgCl2, 2 mm dithiothreitol)

Pure protein fractions were concentrated to approximately

1 mm, 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))1for 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 25C 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 Smin and Smax represent minimum and maximum specific activity, respectively, and n the Hill coefficient:

specific activity¼ Sminþ ðSmax SminÞ: ½protein

n

Knþ ½proteinn ð1Þ

Isothermal titration calorimetry (ITC) All ITC experiments were performed at 25C in buffer C using a Microcal AutoITC200 (GE Healthcare, Munich, Germany) The cell was loaded with 150 lm of protein and was titrated against a 3-fold excess of nucleotide by 20 injection steps of 1.8 lL each using 2.25 mm of each nucle-otide All nucleotides used were purchased from Jena Biosciences (Jena, Germany), the concentration was deter-mined by UV absorption at 254 nm (e = 13 700 (MÆcm))1), and their purity was checked by HPLC analysis (> 98% for all used nucleotides) Data analysis was performed using

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 MgCl2, 150 mm NaCl) contained 200 lm of the nucleotide, and 300 lm AlCl3and

10 mm NaF were added in the case of GDPÆAlFx 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)

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 25C 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

using protein solutions (typically approximately 100 lm)

containing 0.5 lm of the mant-labeled nucleotide to avoid

dilution of the fluorophore The data were analysed using a

quadratic binding equation as described previously [19]

Stopped flow measurements

All measurements were performed using an SFM400

stopped flow apparatus (Bio-Logic, Claix, France) For

association kinetic experiments, 38 lL of a nucleotide

solu-tion (0.5 lm) and 38 lL of hGBP5a⁄ b or hGBP5ta

(increasing concentrations starting from a 10-fold molar

excess) were mixed at 14 mLÆs)1 In the case of mant-GTP

association, it was necessary to use increasing

concentra-tions of the nucleotide because of nucleotide hydrolysis

(0.5 lm of protein was mixed with increasing

concentra-tions of mant-GTP starting from a 15-fold molar excess)

Fluorescence was excited at 295 nm (mant-GTP) or

366 nm (all others), and recorded using a 420 nm cut-off

filter For mant-GTP, we used a fluorescence resonance

energy transfer approach (primary excitation of the

trypto-phans in the G-domain and transfer to the mant-group of

the nucleotide) to minimize excitation of unbound

nucleo-tide and thereby loss of signal quality The traces were

fitted using a single rate constant, and the resulting rates

(kobs) were plotted against the protein or mant-GTP

con-centration Using a linear fit, the association rate constants

were extracted from the slope (kon), and the intercept

rep-resents the dissociation rate (koff) In the case of

displace-ment experidisplace-ments, 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 kdiss

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 Kd= koff⁄ kon

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 37C

Fig S2 Sequence alignment of the hGBP1⁄ 2 ⁄ 5 LG domains

Fig S3 Displacement experiments

This supplementary material can be found in the online version of this article

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