Báo cáo khoa hoc : The guanine cap of human guanylate-binding protein 1 is responsible for dimerization and self-activation of GTP hydrolysis

In this study, we show that the guanine cap of hGBP1 is the key structural element responsible for dimerization, and is thereby essential for self-activation of the GTPase activity.. Stu

responsible for dimerization and self-activation of GTP

hydrolysis

Mark Wehner*, Simone Kunzelmann*, and Christian Herrmann

Ruhr-Universita¨t Bochum, Physikalische Chemie I – AG Proteininteraktionen, Germany

Keywords

dynamin; GTPase; guanine cap;

guanylate-binding protein; interferons

Correspondence

C Herrmann, Ruhr-Universita¨t Bochum,

Physikalische Chemie I, Universita¨tsstr 150,

44780 Bochum, Germany

Fax: +49 2343214785

Tel: +49 2343224173

E-mail: chr.herrmann@rub.de

*These authors contributed equally to this

work

Present address

MRC National Institute for Medical

Research, Division of Physical Biochemistry,

The Ridgeway, Mill Hill, London NW7 1AA,

UK

(Received 19 July 2011, revised 7 October

2011, accepted 27 October 2011)

doi:10.1111/j.1742-4658.2011.08415.x

Human guanylate-binding protein 1 (hGBP1) belongs to the superfamily of large, dynamin-related GTPases The expression of hGBP1 is induced by stimulation with interferons (mainly interferon-c), and it plays a role in dif-ferent cellular responses to inflammatory cytokines, e.g pathogen defence, control of proliferation, and angiogenesis Although other members of the dynamin superfamily show a diversity of cellular functions, they share a common GTPase mechanism that relies on nucleotide-controlled oligomeri-zation and self-activation of the GTPase Previous structural studies on hGBP1 have suggested a mechanism of GTPase and GDPase activity that,

as a critical step, involves dimerization of the large GTP-binding domains

In this study, we show that the guanine cap of hGBP1 is the key structural element responsible for dimerization, and is thereby essential for self-activation of the GTPase activity Studies of concentration-dependent GTP hydrolysis showed that mutations of residues in the guanine cap, in particular Arg240 and Arg244, resulted in higher dissociation constants of the dimer, whereas the maximum hydrolytic activity was largely unaffected Additionally, we identified an intramolecular polar contact (Lys62–Asp255) whose mutation leads to a loss of self-activation capability and controlled oligomer formation We suggest that this contact structurally couples the guanine cap to the switch regions of the GTPase, translating the structural changes that occur upon nucleotide binding to a change in oligomerization and self-activation

Structured digital abstract

l hGBP1 and hGBP1 bind by molecular sieving ( View interaction )

Introduction

A variety of regulation processes in cells depend on

reg-ulatory GTPases [1,2] These include signal

transduc-tion, e.g heterotrimeric G-proteins and members of the

Ras superfamily [3,4], regulation of translation, e.g

EF-Tu [5], and vesicle recycling, e.g dynamin [6,7]

These regulatory GTPases cycle between two different

states, which are established by the bound nucleotide

and the resulting conformation of the protein [8] Usually, the GTP-bound state is the active state of the GTPase, which allows interaction with effector molecules, whereas the GDP-bound state only weakly interacts with effectors [9] By intrinsic or GTPase-activating protein-activated hydrolysis of GTP, result-ing in the GDP-bound state, the GTPase is inactivated

Abbreviations

GBP, guanylate-binding protein; GppNHp, guanosine 5¢-(bc-imino)-triphosphate; hGBP1, human guanylate-binding protein 1; LG, large GTP-binding; mant, N-methylanthraniloyl.

Guanine nucleotide exchange factors accelerate the

exchange of the bound GDP for GTP, and thereby

convey a signal for activation [10]

In contrast to the small GTPases of the Ras

super-family, members of the superfamily of large

dynamin-related GTPases are characterized by high intrinsic

GTPase activity, relatively low nucleotide affinities,

high nucleotide dissociation rates, and self-stimulation

of GTPase activity (reviewed in [7]) This

self-stimula-tion is usually coupled to nucleotide-dependent

oligo-merization and⁄ or lipid binding Members of the

dynamin superfamily have roles in numerous membrane

processes, such as budding, fission and organelle

divi-sion [11,12] Among these dynamin-related proteins, the

guanylate-binding proteins (GBPs) form a family of p67

interferon-inducible GTPases The best characterized

member of this family, human GBP1 (hGBP1), was

found to exhibit antiviral and antiangiogenic activity,

but its cellular function is not yet entirely understood

[13–15] Recent studies have shown involvement of

mur-ine GBPs in defence against intracellular pathogens by

recruiting NADPH oxidase to the pathogen vacuole,

leading to the production of toxic superoxide, which

kills the pathogens [16] Like other dynamins, hGBP1

oligomerizes in a nucleotide-dependent manner and

thereby stimulates GTPase activity [17] Furthermore, it

has been shown that hGBP1 has the unique ability to

hydrolyse GTP not only to GDP but also, in two

successive steps, to GMP, with GMP being the major

product [18–20] Analysis of the structures obtained

from X-ray crystallography suggested that dimer

forma-tion is responsible for self-stimulaforma-tion of GTPase

activ-ity [21] Dimer formation is induced upon GTP binding,

and leads to the reorientation of the catalytic Arg48

and Ser73 towards the active site The ‘arginine finger’

(Arg48) contacts the phosphates to stabilize the negative

charges developing in the transition state [21] The role

of Ser73 is the nucleophilic activation of the attacking

water molecule Mutation of these residues leads to the

loss of self-stimulation, whereas dimer formation after

GTP binding is not impaired [21,22] In this study, we

further address the initial step of self-stimulation, the

dimerization step We investigate the guanine cap’s role

in dimer formation, and we elucidate the coupling of

nucleotide binding and guanine cap reorientation

Results and Discussion

The guanine cap is essential for dimerization of

hGBP1

After investigation of the known structures of the large

GTP-binding (LG) domain of hGBP1 [21], residues

that participate in intermolecular interactions in the dimerization interface were selected for mutagenesis Charged residues were mainly chosen, for reasons explained below These included residues in switch II (Glu105), residues in the guanine cap (Arg240, Arg244, Arg245, and Asp259), and residues following the guanine-binding motif (Ser186 and Asp192) (Fig S1) To identify the residues involved in dimeriza-tion and self-activadimeriza-tion of hydrolysis, we measured GTPase activity as a function of hGBP1 concentration,

as described previously [17,22,23] The mutant proteins were incubated at varying concentrations with a large excess of GTP At different time points, the reaction mixtures were analysed by HPLC to determine the hydrolysis rate Oligomerization-dependent self-activa-tion of hGBP1 is indicated by an increase in specific activity with higher protein concentrations, because, at higher concentrations, a larger fraction will be in an oligomeric state and thus GTP hydrolysis will be faster (Fig 1) As described previously [22–24], data can be analysed with a quadratic binding equation that gives two parameters: Kdimer, the apparent dissociation con-stant of hGBP1 dimers; and smax, the specific activity

at saturating protein concentrations For wild-type hGBP1, these parameters were Kdimer= 0.03 lM and

smax= 0.38 s)1 under low-salt conditions With addi-tion of 200 mMNaCl to the buffer, the apparent dimer dissociation constant was shifted towards 0.68 lM, whereas nucleotide binding and the maximal activity

[hGBP1 or mutant] (µ M )

Fig 1 Concentration-dependent GTP hydrolysis catalyzed by wild-type hGBP1 and the E105A, R240A and R244A mutants The initial rates of GTP turnover were normalized to the protein concen-tration (specific activity) and plotted against the protein concentra-tion The maximal specific activity (smax) and dimer dissociation constant (Kdimer) were obtained from a quadratic equation modelling GTPase stimulation by dimer formation The parameters obtained for the different mutants are summarized in Table 1 WT, wild-type.

were hardly changed (Table 1) This rather pronounced

change in Kdimer suggests that dimerization is strongly

driven by electrostatic interactions Therefore, we

focused mainly on charged residues in the

protein–pro-tein interface for our mutagenesis study

hGBP1 mutants were analysed in the same manner

as the wild-type, and Kdimerand smaxvalues were

deter-mined (Fig 1; Table 1) As with the wild-type, only

almost undetectable GDPase activity was observed for

all mutants studied, and the GMP⁄ GDP product ratio

was similar for the wild-type and all mutants

Muta-tions of Glu105, Asp192 and Ser186 to alanine had

only a minor effect on Kdimer (less than fourfold) In

contrast, mutations of residues located in the guanine

cap (residues 239–259), in particular Arg240 and

Arg244, to alanine yielded a strong increase in Kdimer

(Fig 1) These two mutations, R240A and R244A, led

to 75-fold and 120-fold increases in Kdimer, respectively

(Table 1) Thus, mutation of these guanine cap

argi-nines decreases the ability of hGBP1 to self-stimulate

by weakening the LG dimer interaction as compared

with the wild-type (see also size-exclusion

chromato-graphy) By using Kdimerto calculate the change in free

energy with the Gibbs–Helmholtz equation, it is

possible to estimate the contributions of single residue

contacts to the dimerization indirectly from the

self-activation properties Whereas the whole dimerization

energy derived by this method was  43 kJÆmol)1,

mutations of Arg240 and Arg244 to alanine led to

cal-culated DDG values of 11.0 and 11.5 kJÆmol)1,

respec-tively Combination of these DDG values showed that

these two residues together are responsible for half of

the binding energy of the dimer, emphasizing the

crucial importance of Arg240 and Arg244 for hGBP1

self-activation by dimerization Whereas the changes in

Kdimer caused by these mutations were about two

orders of magnitude, the changes in smax were small, and did not exceed threefold Hence, a high GTP turn-over rate can still be achieved by these mutants if high concentrations of hGBP1 are present to saturate the monomer–dimer equilibrium This indicates that the GTP hydrolysis step itself is not impaired

Mutations of two other guanine cap residues, Arg245 or Asp259, to alanine indicated that each resi-due is responsible for about 10% of the binding energy As described above, mutations outside the guanine cap (D192A, E105A, and S186A) have only a small effect on the binding energy of the dimer (up to 7% for D192A) In addition, two other residues in the switch regions, Glu72 (switch I) and Asn109 (switch II), that form intermolecular contacts in the crystal structure were mutated previously, and they did not show a large difference Kdimer [22] This identifies the guanine cap as the major element responsible for dimer formation in hGBP1

Mutations of the guanine cap do not affect nucleotide binding

Given the fact that the guanine cap forms a hydropho-bic pocket for the guanine base moiety, less self-stimu-lation might be also caused by lower nucleotide binding affinities and not exclusively by decreased dimerization In order to exclude any effects of different nucleotide binding affinities, we performed fluorescence titrations with N-methylanthraniloyl (mant)-labelled nucleotides Representative fluore-scence titrations are shown in Figs 2 and S2, and the obtained dissociation constants are summarized in Table S1 The observed nucleotide dissociation constants showed only marginal changes as compared with those for the wild-type reported earlier [19,22,23]

As for the wild-type, the relative binding affinities were GMP > guanosine 5¢-(bc-imino)-triphosphate (GppNHp) > GDP Our original assumption of an exclusive effect on dimerization is strongly supported

by these observations of similar nucleotide-binding properties

Dimerization at the LG domains is inhibited, but tetramers are still formed

After analysing the concentration-dependent self-stim-ulation of the mutants, as shown above, we were inter-ested in the ability of hGBP1 to form oligomers in complex with various nucleotides and nucleotide analogues The wild-type has been shown to form dimers in complex with the GTP analogue GppNHp via an interaction of the LG domains [17,21] In recent

Table 1 Parameters of GTP hydrolysis obtained by

concentration-dependent hydrolysis at 25 C (constants as defined in the text).

ND, not determined; WT, wild-type.

Mutation

Kdimer (l M )

smax (s)1)

DDG (kJÆmol)1)

studies, we have identified a second interaction

interface at the helical domain, which is accessible only

as a result of GTP hydrolysis [25,26] This leads to the

formation of tetramers, which can be trapped by the

complex of GDP and aluminium fluoride [17,25,26]

Thus, we used size-exclusion chromatography to

inves-tigate whether contact formation of two LG domains

and of two helical domains occur independently

To directly analyse the oligomerization behaviour of

the mutants, we performed analytical gel filtration

experiments In agreement with the hydrolysis data, we

observed that mutants with strongly increased Kdimer

values did not form or only partly formed dimers in

presence of GppNHp (Figs 3 and S3; Table 2) With a

protein concentration (20 lM) that saturates dimer

for-mation of the wild-type, no sign of dimer forfor-mation

was observed with the R240A mutant The R244A, R245A and D259A mutants showed increasing frac-tions of a dimeric species, but > 90% of the protein was in a monomeric state The D192A and E105A mutants exhibited very similar behaviour to the wild-type These mutants formed mainly dimers, and only a very small fraction of monomeric protein was observed, in good agreement with the marginal inhibi-tion of self-stimulainhibi-tion in concentrainhibi-tion-dependent GTPase activity Thus, the GppNHp-dependent dimer-ization reflects the results of self-activation obtained by GTP hydrolysis experiments, and directly proves the involvement of Arg240 and Arg244 in dimer formation

of hGBP1

As described above, hGBP1 forms tetramers in the

‘trapped state’ of GTP hydrolysis with GDP alumin-ium fluoride [22,23] All mutants that showed only minor effects on self-activated hydrolysis and dimeriza-tion were also able to form tetramers in complex with GDP aluminium fluoride, similar to the wild-type (Tables 2 and S2) Surprisingly, proteins with muta-tions that significantly impaired self-stimulation and dimerization were also able to form tetramers in the presence of GDP aluminium fluoride In contrast to the wild-type, these mutants showed an elution profile corresponding to the tetramer and, partially, the monomer (Fig 3) The ability of the R240A and R244A mutants to form higher oligomers, even though the dimerization was strongly impaired, can be

[hGBP1 WT] (µM)

[hGBP1 D255A] (µM)

Fig 2 Representative mant-nucleotide binding experiment with

wild-type hGBP1 (upper panel) or the hGBP1 mutant D255A (lower

panel) A solution containing 0.5 l M mant-GMP (circles), mant-GDP

(triangles) or mant-GppNHp (squares) was titrated with wild-type

hGBP1 and the D255A mutant, respectively, and the observed

fluo-rescence was normalized to the fluofluo-rescence of the nucleotide

alone The obtained nucleotide dissociation constants of all mutants

used in this study are summarized in Table S1 WT, wild-type.

Fig 3 Size-exclusion chromatography experiment with wild-type hGBP1 (upper panel) or the guanine cap mutant R240A (lower panel) in the nucleotide-free (dotted lines), GppNHp (solid black line) and GDP aluminium fluoride (grey line) states The molecular masses of standard proteins are indicated by arrows Elution of all proteins was followed by absorbance at 280 nm, and the elution volume (V e ) was normalized to the exclusion volume (V 0 ) The results for other mutants used in this study are summarized in Table 2 and in more detail in Table S2 WT, wild-type.

explained by the presence of two independent

interac-tion sites, as described above In the case of the

wild-type, the first dimerization at the LG domains

occurs as a result of GTP binding In the course of

hydrolysis, the secondary interface becomes accessible,

and additional interaction at the C-terminal part

occurs, resulting in the formation of a tetramer, i.e a

dimer of a dimer In contrast to the behaviour of the

wild-type, mutations of the LG dimer interface weaken

the first dimerization step, but the mutants are still

able to form the contact at the secondary interface

This interaction of the C-terminal parts of hGBP1

results in two weak dimerization sites in proximity,

facil-itating tetramer formation

The guanine cap indirectly senses the bound

nucleotide

By analysing the guanine cap conformation in different

nucleotide states, we found that, in the GMP-bound

state of the LG domain, the guanine cap has an ‘open’

conformation, which does not allow dimerization (Fig 4) X-ray structures of GTP-bound or GDP-bound hGBP1 are not available, as these nucleotides are hydrolysed The hGBP1 structures of the trapped states in complex with one of the analogues, GppNHp, GDP aluminium fluoride, or GMP aluminium fluoride, show the guanine cap in a ‘closed’ conformation that facilitates dimer formation of two LG domains Thus, there is a relationship between the guanine cap confor-mation and the presence of a b-phosphate Detailed analysis of the LG domain X-ray structures reveals an intramolecular electrostatic contact between the guan-ine cap Asp255 and Lys62 located in the region between the P loop (residues 45–52) and switch I (resi-dues 65–77) This contact is found in the GTP ana-logue structures but is lost in the GMP-bound LG domain, leading to the hypothesis that it could be responsible for the ‘closed’ guanine cap conformation resulting in dimer formation and, subsequently, GTPase stimulation It was shown earlier that GTP binding and not hydrolysis, i.e binding of nonhydroly-sable GppNHp, results in dimer formation of hGBP1 Thus, the loss of the Asp255–Lys62 contact should result in loss of self-stimulation capability, and the GTPase activity should be at a low level and concen-tration-independent; that is, only the unstimulated monomer activity should be observed Indeed, after introducing an alanine mutation at either side of the contact (K62A or D255A), we found a nearly constant specific activity at the unstimulated level of GTP turn-over turn-over three orders of magnitude of hGBP1 concen-tration (Fig 5) By fluorescence ticoncen-tration, we showed that the nucleotide binding is similar to that of the wild-type, indicating that the effect on hydrolysis is not caused by changed nucleotide binding Using size-exclusion chromatography, we found the oligomer

Table 2 Oligomerization of hGBP1 mutants WT, wild-type.

Nucleotide-free GppNHp GDP AlF x

‡ 200 kDa

Oligomer

‡ 200 kDa

Oligomer

‡ 200 kDa

D255A Oligomer

‡ 200 kDa

Oligomer

‡ 200 kDa

Oligomer

‡ 200 kDa

Fig 4 Conformations of hGBP1’s guanine cap in different nucleotide states (A) Structural overview of the GppNHp-bound hGBP1 LG domain dimer [Protein Data Bank (PDB) 2bc9 [20]] The two monomers (blue and green) are facing each other, and the guanine caps are highlighted in purple or yellow (B) The electrostatic contact Lys62–Asp255 is lost when GMP is bound, and the guanine cap adopts a relaxed conformation that does not support dimer formation The colours of the protein chain used are blue for GppNHp (PDB 2bc9 ) and white for GMP (PDB 2d4h ).

state of both the K62A and D255A mutants to be

independent of the bound nucleotide Control of

defined hGBP1 oligomer formation seems to be lost,

because, with every nucleotide or nucleotide analogue

used, we observed similar elution profiles, showing

oligomers with molecular masses > 200 kDa

corre-sponding to a size of more than a tetramer This

demonstrates that the contact between Lys62 and

Asp255 or Ala255 is essential for the control of

oligomer formation by the nucleotide state We

conclude that the contact between Lys62, in proximity

to the essential switch I Thr75, and the guanine cap

Asp255 is responsible for the switch-like character of

the guanine cap, and is of crucial importance for the

establishment of the guanine cap conformation As a

result of this active conformation, the LG domains are

able to dimerize, being mainly stabilized by Arg240

and Arg244 This leads to the rearrangement of the

catalytically important residues, such as Arg48 and

Ser72 [21], and thereby self-stimulation of GTPase

activity

Concluding remarks

Previous studies on the oligomerization of hGBP1

focused on the role of dimerization in GMP

produc-tion [27] and dimer formaproduc-tion in living cells [28]

How-ever, the crucial residue positions for dimerization and

the resulting self-activation remain elusive In this

study, we show the importance of hGBP1’s guanine

cap for the self-stimulation of GTPase activity in

solu-tion, which was shown to be similar to GTPase

activ-ity of fully modified hGBP1 bound on lipids [29] By

mutagenesis, we were able to identify Arg240 and Arg244 as the major determinants of GTP-induced dimerization, which, in turn, leads to efficient self-stim-ulation of the GTPase activity Using the apparent dimerization constant, we were able to estimate the rel-ative contributions of single residues to dimerization, and this showed that about 50% of the energy is attributable to the guanine cap residues 240 and 244

By further analysis of the guanine cap conformation in the published crystal structures, an electrostatic contact between the guanine cap (Asp255) and the switch I region (Lys62) was identified Mutation of the residues

in this contact completely abolished self-activation of hGBP1 We conclude that, after GTP binding to the

LG domain, the contact between switch I and the guanine cap is formed, which ‘transmits’ the informa-tion about the nucleotide state to the guanine cap As

a result, the guanine adopts its ‘active’ conformation and the LG domains form a dimer This dimerization leads to the well-described activation of GTP hydroly-sis by rearrangement of the catalytically important residues Arg48 and Ser72 The results presented in this study give insights into the early processes of hGBP1’s self-stimulation, and the mutations characterized might

be valuable tools for further understanding hGBP1’s cellular function

Experimental procedures Site-directed mutagenesis and protein purification

Wild-type hGBP1 and the mutants used in this study were cloned into pQE80L expression vectors (Qiagen, Hildes-heim, Germany), expressed in Escherichia coli BL21(DE3), and purified as described previously [19] Mutations were

(Stratagene, Amsterdam, The Netherlands), according to the manufacturer’s instructions All introduced mutations were verified by DNA sequencing with a 3130xl sequencer (Applied Biosystems, Foster City, CA, USA) Concentra-tions of the purified proteins were measured by UV absorp-tion at 276 nm [e276 nm= 45 400 (MÆcm))1] [19]

Hydrolysis assay

Hydrolysis assays were performed as described previously

Germany) and different concentrations of hGBP1 in

GTP ensures near complete saturation of the protein molecules with nucleotide Aliquots were injected onto a

[hGBP1 or mutant] (µ M )

Fig 5 Concentration-dependent GTP hydrolysis of wild-type

hGBP1 (solid squares) and the K62A (filled circles) and D255A

(empty triangles) mutants Data were treated similarly to the data

shown in Fig 1.

Chromolith RP18e HPLC column (Merck, Darmstadt,

1.25% (v⁄ v) acetonitrile], and absorption was analysed at

254 nm with a MD-2015 diode array detector (Jasco,

Gross-Umstadt, Germany) The initial (linear) phase of

steady-state hydrolysis (remaining GTP > 60%) was

analy-sed by linear regression, and the resulting rates were

nor-malized to the protein concentration, yielding the specific

activity Data were analysed as described previously, with

a quadratic binding equation that gives two parameters:

Kdimer, the apparent dissociation constant of hGBP1

dimers, and smax, the specific activity at saturating protein

concentrations [22–24]

Size-exclusion chromatography

Analytical gel filtration experiments were performed with a

Superdex 200 10⁄ 300 (GE Healthcare, Mu¨nchen, Germany)

pH 7.9, 5 mM MgCl2, and 2 mM dithiothreitol) contained

200 lMof the nucleotide and, in the case of GDP⁄ GMP

alu-minium fluoride, additionally 300 lMAlCl3and 10 mMNaF

Protein at a concentration of 20 lMwas preincubated in the

elution buffer for 5 min on ice prior to injection Size

calibra-tion was carried out using standard proteins with molecular

masses between 29 kDa and 200 kDa (the corresponding

elution volumes are marked by arrows in the plots) The void

volume (V0) was measured with the use of Blue Dextran

Elution was followed by monitoring the absorbance at

280 nm with an MD2015 diode array detector (Jasco)

Fluorescence titrations

Kontron SFM25 fluorospectrometer (Kontron, Zu¨rich,

Switzerland) and 2¢ ⁄ 3¢-mant-labelled nucleotides (Jena

Bioscience, Jena, Germany) The excitation and emission

wavelengths were 366 nm and 435 nm, respectively

Mant-labelled nucleotide (0.5 lM) was titrated with protein

solu-tions (typically 100 lM) containing 0.5 lM mant-labelled

nucleotide The data were analysed with a quadratic

binding equation as described previously [19,22,23]

Acknowledgement

This work was financially supported by Deutsche

Forschungsgemeinschaft (DFG)

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Supporting information The following supplementary material is available: Fig S1 Positions of the mutated residues

Fig S2 Mant-nucleotide binding experiments

Fig S3 Size-exclusion chromatography experiments Table S1 Dissociation constants of hGBP1 in complex with various mant-nucleotides at 25C

Table S2 Evaluation of the size-exclusion chromatog-raphy runs

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