The activation mechanism of Irga6, an interferon-inducible GTPase contributing to mouse resistance against Toxoplasma gondii
- Nikolaus Pawlowski1,
- Aliaksandr Khaminets†1, 3,
- Julia P Hunn†1,
- Natasa Papic1, 4,
- Andreas Schmidt1, 5,
- Revathy C Uthaiah1, 6,
- Rita Lange1,
- Gabriela Vopper1,
- Sascha Martens1, 7,
- Eva Wolf2, 8 and
- Jonathan C Howard1Email author
© Pawlowski et al; licensee BioMed Central Ltd. 2011
Received: 14 October 2010
Accepted: 28 January 2011
Published: 28 January 2011
The interferon-inducible immunity-related GTPases (IRG proteins/p47 GTPases) are a distinctive family of GTPases that function as powerful cell-autonomous resistance factors. The IRG protein, Irga6 (IIGP1), participates in the disruption of the vacuolar membrane surrounding the intracellular parasite, Toxoplasma gondii, through which it communicates with its cellular hosts. Some aspects of the protein's behaviour have suggested a dynamin-like molecular mode of action, in that the energy released by GTP hydrolysis is transduced into mechanical work that results in deformation and ultimately rupture of the vacuolar membrane.
Irga6 forms GTP-dependent oligomers in vitro and thereby activates hydrolysis of the GTP substrate. In this study we define the catalytic G-domain interface by mutagenesis and present a structural model, of how GTP hydrolysis is activated in Irga6 complexes, based on the substrate-twinning reaction mechanism of the signal recognition particle (SRP) and its receptor (SRα). In conformity with this model, we show that the bound nucleotide is part of the catalytic interface and that the 3'hydroxyl of the GTP ribose bound to each subunit is essential for trans-activation of hydrolysis of the GTP bound to the other subunit. We show that both positive and negative regulatory interactions between IRG proteins occur via the catalytic interface. Furthermore, mutations that disrupt the catalytic interface also prevent Irga6 from accumulating on the parasitophorous vacuole membrane of T. gondii, showing that GTP-dependent Irga6 activation is an essential component of the resistance mechanism.
The catalytic interface of Irga6 defined in the present experiments can probably be used as a paradigm for the nucleotide-dependent interactions of all members of the large family of IRG GTPases, both activating and regulatory. Understanding the activation mechanism of Irga6 will help to explain the mechanism by which IRG proteins exercise their resistance function. We find no support from sequence or G-domain structure for the idea that IRG proteins and the SRP GTPases have a common phylogenetic origin. It therefore seems probable, if surprising, that the substrate-assisted catalytic mechanism has been independently evolved in the two protein families.
Immunity-related GTPases (IRG proteins/p47 GTPases) are major contributors to cell autonomous resistance against the intracellular protozoal pathogen, Toxoplasma gondii [1–3]. For nomenclature of IRG proteins, see Methods and . Multiple members of the family are expressed in cells induced by interferon-γ (IFNγ). Many IRG proteins, including Irga6 (IIGP1) and Irgb6 (TGTP) relocate from resting cytoplasmic compartments to the parasitophorous vacuole membrane (PVM) of avirulent T. gondii [1, 5, 6]. Loading of IRG proteins onto the T. gondii PVM is followed by vesiculation and rupture of the PVM and death of the parasite [5, 7, 8]. Irga6 at the PVM is in the active, GTP-bound state, while cytoplasmic Irga6 is inactive and probably GDP-bound [9, 10].
Irga6 forms GTP-dependent oligomeric complexes in vitro and in vivo and hydrolysis of the GTP substrate is cooperatively activated [10, 11]. These enzymatic properties of Irga6 together with the relatively high molecular mass of 47 kDa and the nucleotide binding affinities in the micromolar range  are also found in several other families of large GTPases, including members of the dynamin superfamily , associated with membrane remodelling and, like Mx proteins, resistance against intracellular pathogens [1, 13].
The structure of the Irga6 protein was determined some years ago . The protein consists of a Ras-like G-domain  and a helical domain (Additional file 1). The G-domain contains three conserved GTP-binding motifs (G1, G3 and G4)  and two flexible switch regions, switch I and switch II . Homology considerations suggest that the structure of Irga6 can provide a reasonable template for the IRG family. Three members of the IRG family, Irgm1 (LRG-47), Irgm2 (GTPI) and Irgm3 (IGTP), carry a unique substitution of the otherwise universally conserved P-loop (G1 motif) lysine (GKS subfamily) to methionine (GMS subfamily) (Additional file 2) [4, 18]. In the absence of GDP-dependent negative regulatory interactions with the three GMS proteins, GKS subfamily members including Irga6 activate prematurely in the cytoplasm, form GTP-dependent aggregates, and are unable to accumulate on the PVM of invading T. gondii [9, 10, 19].
Little is known about the relationship between the GTP-dependent activation of Irga6 and pathogen resistance. Our study poses some specific questions directed towards an understanding of these processes at a molecular level: where are the interfaces that participate in oligomerisation and interactions with other IRG proteins, how is GTP hydrolysis activated in the oligomeric complexes, and finally, is oligomeric complex formation required for resistance against T. gondii? We carried out an extensive mutagenesis screen to address the first question and found a novel interface of Irga6 located in the G-domain. This interface is required for oligomerisation and for accelerated hydrolysis of GTP. From experimental analysis of this interface we can propose a structural model for the activation of GTP hydrolysis that is, surprisingly, based on the hydrolytic mechanism of the signal recognition particle (SRP) and its receptor (SRα) [20, 21]. We demonstrate that the catalytic interface includes the bound GTP substrate and that the 3'hydroxyl (3'OH) of the nucleotide ribose is required for activation of hydrolysis in trans. We also show the engagement of the catalytic interface in both the activating interaction of Irga6 with Irgb6 [6, 9] and the inhibitory interaction between Irga6 and the GMS subfamily protein, Irgm3 . Lastly, we show that the integrity of the catalytic interface of Irga6 is required for the accumulation of the active, GTP-bound protein at the T. gondii PVM.
The catalytic interface is localised on the G-domain
Mutations of the residues Arg31, Lys32, Lys169, Lys176, Arg210 and Lys246 (Figure 1, yellow) also reduced GTP-dependent oligomerisation to some extent (Additional file 3), but none completely (compare Additional file 4 and 5). These residues formed a loosely defined "secondary patch" on the Irga6 surface (Figure 1d, f). Unlike the catalytic interface, however, the secondary patch is interspersed with residues which, when mutated, had no effect on oligomerisation, and indeed a substantial part of the secondary patch area could be replaced simultaneously without preventing oligomerisation (data not shown). At present, therefore, we do not consider the secondary patch to be an oligomerisation interface. The oligomerisation of Irga6 was not prevented by numerous other mutations (Figure 1, green), suggesting the absence of a second well-defined surface interface contributing to oligomerisation.
The majority of catalytic interface mutants including T102A and T108A  had no significant effect on the binding affinity for GTP (Additional file 6). Thus the failure of these mutants to oligomerise is not caused by reduced nucleotide binding. The mutations E77A, R159E, K161E and N191R slightly decreased the nucleotide binding affinity (Additional file 6) but it is unlikely that this caused the loss of oligomerisation because the G4-motif mutant, Irga6-D186N, with a considerably lower binding affinity for guanine nucleotides, oligomerised relatively efficiently in the presence of GTP (see below). Furthermore, none of the mutants of the secondary patch, which all showed reduced nucleotide-binding affinities (Additional file 6), prevented oligomerisation of Irga6.
Irga6 crystallizes as a rotationally symmetrical dimer  (Additional file 7). Mutants of the crystal dimer interface were seen to oligomerise less efficiently than the wild-type (WT) and it was suggested that this interface might participate in cooperative GTP-dependent activation . The crystal dimer interface does not obstruct the catalytic interface described here (Additional file 8) and could therefore contribute to active Irga6 oligomerisation. Mutants of the four crystal dimer interface residues Leu44, Lys48, Ser172 and Met173, that had been examined earlier , were therefore re-assayed (Additional file 9). Mutants of nine further residues (Glu37, Glu43, Glu142, Lys169, Lys175, Lys176, Glu177, Arg218 and Glu224) in the crystal dimer interface were also analysed (Additional file 3). Under the conditions of these experiments, which were more stringent than those used previously, out of 13 residues mutated in the crystal dimer interface only the mutations of Lys169 and Lys176, two residues already identified in the secondary patch, partially inhibited oligomerisation (Additional file 7). Furthermore, the formation of the crystal dimer is not nucleotide-dependent , whereas the oligomerisation of Irga6 requires GTP binding . These arguments urge that the crystal dimer interface does not identify the oligomerisation interface associated with activation. Thus no convincing second interface required for oligomerisation has yet been found on the surface of the known crystal structure  of Irga6 (Figure 1), suggesting that oligomerisation requires a cryptic interface exposed following GTP binding or dimer formation at the catalytic interface.
The catalytic interaction of the SRP GTPases provides a scaffold for a model of the Irga6 dimer
The ribose of the bound nucleotide is part of the catalytic interface
The base of bound nucleotide is part of the catalytic interface
The nucleotide-binding preference of Irga6 was changed from guanine to xanthine based nucleotides by the corresponding G4-motif mutation D186N (Additional file 14). Unexpectedly, despite a nine-fold higher affinity for XTP than for GTP, Irga6-D186N hydrolysed GTP more efficiently than XTP (Additional file 15). Oligomerisation of Irga6-D186N (Figure 6c) accompanied by GTPase activity (Figure 6d) could be activated by GTP, albeit inefficiently, and both were abolished when the high affinity ligand, XTP, was added at a concentration 1/10 that of GTP (Figure 6c, d). This shows that the replacement of the surface exposed C2 amino-group, of bound GTP, by the oxo-group, of XTP, inhibits Irga6 oligomerisation, implicating the nucleotide base as part of the interaction interface between the complex-forming molecules, as in Ffh-FtsY. In the dimer model the two relatively close trans neighbours of the GTP base C2 amino-group are Glu77 and Ser132 (Figure 6a). Consistently, mutations of Glu77 and Ser132 both caused loss of oligomerisation (Figure 2a).
The 3'OH of the GTP ribose is required for trans-activation of GTP hydrolysis
For the Ffh-FtsY heterodimer the essential activation function of the 3'OH is mediated in trans . We, therefore, investigated whether the basal hydrolysis of radioactively labeled 3'dGTP could be enhanced by addition of unlabeled GTP, 2'dGTP, 3'dGTP or 2'3'ddGTP. Since each Irga6 monomer has only one nucleotide-binding site, an increase in 3'dGTP hydrolysis by addition of GTP must be due to an activation by a second, GTP-loaded, monomer in trans. Furthermore, trans-activation of hydrolysis of 3'dGTP, a nucleotide which itself does not contain the 3'OH, would show the dispensability of the 3'OH in cis. Consistent with the Irga6 dimer model the addition of GTP and 2'dGTP stimulated the hydrolysis of labeled 3'dGTP, whereas the addition of 3'dGTP and 2'3'ddGTP had an inhibitory effect (Figure 7c). Therefore, the 3'OH is required in trans but not in cis for the activation of hydrolysis. A model of the dimer interaction responsible for the trans activation of hydrolysis of labeled 3'dGTP by unlabeled GTP is shown in Figure 7d.
Glu106 is a key residue crucial for the activation of catalysis
Glu106 is part of the flexible switch I region which undergoes nucleotide-dependent conformational changes . In the GDP state Glu106 is exposed and points away from the bound nucleotide, a spatial arrangement that is incompatible with the formation of the dimer as suggested by the model (Figure 8b). However, in the GppNHp state Glu106 can be reoriented towards the γ-phosphate of the bound nucleotide . The GTP ribose 3'OH may stabilize the Glu106 residue in trans in a conformation allowing complex formation and in an orientation required for activation of the catalytic water molecule in cis (Figure 8a). This could initiate a nucleophilic attack on the γ-phosphate and activate GTP hydrolysis.
The catalytic interface of Irga6 is essential for heteromeric interactions between IRG members
The three GMS proteins, Irgm1, Irgm2 and Irgm3, are essential negative regulators of Irga6 . For Irgm3 this interaction has been shown to be GDP-dependent and inhibited by GTPγS . In cells, in the absence of this interaction, Irga6 binds GTP, activates spontaneously, and cannot accumulate on PVMs of invading T. gondii [9, 10]. We were able to confirm the previously documented GDP-dependent interaction between IFNγ-induced Irgm3 and recombinant Irga6 in a pull-down assay, and additionally showed that no interaction occurred when mant-GDP was used (Figure 9b), hinting at usage of the catalytic interface. This was confirmed when two of four mutants of the catalytic interface also blocked the GDP-dependent interaction of Irgm3 with Irga6 (Figure 9b). The two residues whose mutation did not interfere with Irga6-Irgm3 interaction, Gly103 and Ser132, are located in a different part of the catalytic interface from Arg159 and Asn191. These results suggest that the GDP-dependent negative regulatory interaction between Irgm3 and Irga6 indeed involves the catalytic interface, but with a slightly different orientation or a higher affinity from that of the GTP-dependent activating interaction.
The catalytic interface is required for recruitment of Irga6 to the T. gondiiPVM
Irga6 forms GTP-dependent oligomers and GTP hydrolysis is activated in this state . The present study has identified a new catalytic interface (Figure 1) required for the formation of Irga6 oligomers. This interface provides a platform for both positive and negative nucleotide-dependent regulatory interactions between Irga6 molecules and other members of the IRG protein family (Figure 9). These interactions are essential for the activity of the IRG proteins in resistance to T. gondii (Figure 10) . The revealed surface is part of the G-domain, including the nucleotide-binding site and the switch regions (Figure 1). The nucleotide itself is part of the interface (Figures 5 and 6). Structural and biochemical features common to the SRP GTPases and Irga6 suggested a model (Figure 3) for the Irga6 dimer based on the relative orientation of the two nucleotides buried in the SRP-SRα complex [20, 21]. The mutagenesis data were consistent with the proposed model (Figure 4), but the key to the activation of GTP hydrolysis by SRP and SRα in the dimeric complex is the reciprocal trans interaction between the 3'OH of the GTP ribose and the γ-phosphate of the two nucleotides [20, 21]. In strong support for the validity of the SRP-SRα based model of the Irga6 dimer, the 3'OH of the GTP ribose proved to be absolutely required for oligomerisation and GTP hydrolysis by Irga6 and, as in SRP-SRα, this function was exercised in trans only (Figure 7).
Functionally, Irga6 seems closer to the dynamins in that it is involved in the vesiculation and disruption of the PVM , yet the catalytic geometry appears far closer to the SRP GTPases. Despite this distinctive similarity, however, the IRG and SRP protein families appear to be completely unrelated to each other in sequence in those parts of the molecule that compose the catalytic interface (Additional file 19) and belong, in fact, to the two different major clades (SIMIBI and TRAFAC) that have been defined over multiple GTPase families . If indeed, IRG proteins share the unusual catalytic mechanism of SRP [20, 21] then these proteins appear to represent convergent approaches to the same solution. For the SRP GTPases it is clear that the solution is ancient, but until a convincing ancestry for the IRG proteins is found it is not possible to say whether their organization is ancient or derived.
Alternative Irga6 dimer models
So far, the majority of dimeric GTPases for which structure is known engage the two monomers in a parallel orientation and the two nucleotides are separated and do not interact [1, 30, 31]. In contrast, the SRP-SRα paired GTPases engage the two monomers in an anti-parallel orientation with the two nucleotides in reciprocal atomic contact [20, 21]. We explored the feasibility of alternative models of the Irga6 dimer based on the relative orientation of the nucleotides, and consequently of the G-domains, found in the dimeric structures of other GTPases and related ATPases (data not shown). None provided a satisfactory basis on which to explain the properties of Irga6. In models based on EHD2 , MeaB  and MnmE , the two Irga6 G-domains interact via different surfaces that do not include the bound nucleotides. The models based on SEPT2 , GIMAP2 , BDLP , Toc34  and Soj  engage small parts of the catalytic interface in limited interfaces. The catalytic interface is involved in the models based on HypB  and Av2 . The model based on hGBP1  involves the catalytic interface but the subunits overlap in the contact area. The dynamin  based model would involve the catalytic interface, if the subunits were closer. However, none of the alternative models engaging the catalytic interface bring the two nucleotides into atomic contact. None of the models of the Irga6 dimer except that based on SRP-SRα offer an explanation for the critical requirement in trans of the 3'hydroxyl of the GTP ribose for the activation of catalysis (Figure 7c).
The activation of GTP hydrolysis in Irga6 complexes
GAPs work by supplementation of missing catalytic residues (arginine finger; asparagine thumb), and by reorientation and stabilization of the catalytic machinery which is already present in the target protein [44–46]. The model of the Irga6 dimer suggests that the switch regions are stabilized by the interaction of the two Irga6 molecules. In particular, the model suggests that Glu106 (switch I) is stabilized by the trans interaction with the 3'OH of the GTP ribose (Figure 8a). Mutational analysis of Glu106 (Figure 8c, d) together with structural data  urge that this residue activates the catalytic water molecule for the nucleophilic attack on the γ-phosphate in cis and is therefore crucial for the activation of GTP hydrolysis. The finding that the 3'OH of the GTP ribose is essential for activation of GTP hydrolysis in trans (Figure 7c) is consistent with the anticipated function of Glu106.
On complex formation between Ffh and FtsY catalytic residues of the switch I region become reoriented and facilitate GTP hydrolysis in cis. It is proposed that aspartates activate the catalytic water molecules, and that arginines coordinate the γ-phosphates [20, 21, 47]. In contrast to the Irga6 dimer model, there is no trans interaction between the 3'OHs and the catalytic aspartates in the Ffh-FtsY complex; the catalytic aspartates approach the γ-phosphates from a different direction [20, 21]. It may be relevant that acidic residues have been implicated in activating the catalytic water in further dimer-forming GTPases, MeaB , MnmE  and HypB , as also in related ATPases, Soj  and Av2 .
The Irga6 dimer model does not suggest any positively-charged residue that could fulfill an arginine finger-like function. The mutation of the most promising candidate, Lys101 in switch I, to glutamate had no effect on complex formation or GTP hydrolysis (Figure 2). The non-necessity of an arginine finger-like residue was demonstrated for Ran and Rap; instead, in both cases a tyrosine OH was found to interact with the γ-phosphate, and, in the case of Ran, also with the catalytic glutamine [48, 49]. These interactions recall the proposed trans interactions of the 3'OH of the GTP ribose with the γ-phosphate and Glu106 in the Irga6 dimer model (Figure 8a). Generally, the transition state in Irga6 could be stabilized in cis and in trans by hydrogen bond donation from the residues surrounding the nucleotide and also from water molecules that, by analogy to Ffh-FtsY [20, 21], potentially bridge the two opposed nucleotides.
The catalytic interface - a general interaction platform involved in activation and regulation
The catalytic interface is the most conserved part of the Irga6 surface (Additional file 20). It probably represents a central platform engaged in functional interactions between IRG proteins in general. Heteromeric interactions between Irga6 and other members of the IRG family play important regulatory roles in the biological action of Irga6. While Irgb6 enhances the accumulation of activated Irga6  on the T. gondii PVM [6, 9], Irgm3 prevents the premature activation of Irga6 prior to infection by locking the GDP-bound state of the protein . In vitro, the catalytic interface is involved in the GTP-dependent Irga6-Irgb6 interaction (Figure 9a) and also in the GDP-dependent Irga6-Irgm3 interaction (Figure 9b). These results show that the negative regulatory interaction between Irga6 and Irgm3 occurs, like the activating Irga6-Irga6 and Irga6-Irgb6 interactions, via the catalytic interface. The outcome of the Irga6-Irgm3 interaction thus resembles the primary action of a GDP dissociation inhibitor (GDI) . Thus two different functions (GAP and GDI) seem to be mediated through the catalytic interface. All tested mutants of the catalytic interface prevented the Irga6-Irgb6 interaction (Figure 9a), but the Irga6-Irgm3 interaction was prevented only by a subset of the mutants (Figure 9b) suggesting a distinct mode of interaction. The catalytic interface of the GMS proteins, including Irgm3, contains specific substitutions (Additional file 2). The otherwise conserved residues Glu106, Asp164 and Arg159, which are crucial for oligomerisation and GTP hydrolysis, are substituted by arginine, histidine and glutamine respectively in the GMS proteins. The corresponding mutations, E106R, D164H and R159Q in Irga6 have deleterious effects on GTP-dependent complex formation and hydrolysis activation (Figure 8 and Additional file 11). The specific modifications of the catalytic interface in GMS proteins may facilitate complex formation with GDP-bound GKS proteins, thus prolonging their inactive state in the absence of infection.
The catalytic interface plays a central role in the antimicrobial function and is a target for a T. gondiivirulence factor
Upon infection with avirulent T. gondii Irga6 accumulates at the PVM and participates in disruption of the PVM and killing of the parasite [5, 8]. The accumulation of IRG proteins at the PVM is a prerequisite for the antimicrobial function [5, 6, 8]. The biological importance of complexes formed via the catalytic interface is shown by the fact that mutations of this surface strongly diminish the accumulation of Irga6 on the PVM of avirulent T. gondii (Figure 10). Irga6, with other IRG proteins, does not accumulate normally on the PVM of virulent T. gondii and the parasites survive and continue to replicate [6, 8, 50]. The secreted ROP18 kinase  is a major virulence factor of T. gondii . The significance of the catalytic interface for the function of Irga6 is highlighted by the recent finding that the virulence-associated ROP18 kinase from virulent, but not avirulent, T. gondii strains phosphorylates conserved threonine residues, Thr102 and Thr108, in switch I within the catalytic interface of Irga6, thus blocking oligomerisation, GTPase activity and the accumulation of Irga6 at the PVM .
An intracellular way of life can protect pathogens from antibodies, but hosts can deploy other specialized defense mechanisms against such pathogens. Toxoplasma gondii, is an intracellular protozoal pathogen of mammals and birds, and commonly infects humans. Mice exploit a specialized intracellular resistance system, the immunity-related GTPases (IRG proteins), for defense against T. gondii. The IRG protein, Irga6, accumulates rapidly on the membrane surrounding intracellular parasites. Shortly after, this membrane ruptures and the parasite dies. The enzymatic activity, required for the antimicrobial function, of Irga6 is activated in oligomeric complexes formed by the protein.
We define one of the contact surfaces involved in Irga6 oligomerisation, the so-called catalytic interface, which is a part of the G-domain and to which the bound nucleotide contributes. This strongly conserved interface participates in the positive and negative regulatory interactions of Irga6 with Irgb6 and Irgm3 respectively, thus it is a universal platform engaged in interactions between and regulation of IRG proteins. The catalytic interface is essential for the accumulation of Irga6 on the membrane, surrounding T. gondii within infected cells, and is therefore required for the antimicrobial function of the protein.
Further, we propose a model for the dimer formed via the catalytic interface of Irga6, based on the unique substrate geometry and catalytic machinery found in the dimeric complex of the signal recognition particle, Ffh (SRP54), and its receptor, FtsY (SRα). The reciprocal catalytic interaction, made in trans by the 3'hydroxyl of the bound nucleotide ribose, determines the relative orientation of the signal recognition particle and its receptor in the dimeric complex. The 3'hydroxyl of the nucleotide ribose is also essential for Irga6 complex formation and activation of GTP hydrolysis in trans. The model also explains how a catalytic glutamate residue is engaged in the activation of catalysis.
Since there is no distinctive sequence homology between the SRP GTPases and Irga6, we consider that the functional similarity between these two GTPase families is probably the result of convergent evolution.
Nomenclature of IRG proteins
Irga6, the main subject of this study was originally named IIGP [18, 53]. The name was later modified to IIGP1 and biochemical  and structural  studies on the protein were performed under this name. The nomenclature of the whole protein family was rationalized in 2005 under the generic name IRG (immunity-related GTPases) to accommodate its genomic structure and phylogenetic complexity , and IIGP1 was renamed Irga6.
Expression constructs and mutagenesis
Expression constructs were generated by site directed mutagenesis in pGEX-4T-2-Irga6  and pGW1H-Irga6-cTag1  using the QuickChange protocol (Stratagene, La Jolla, CA, USA). Primers used (including reverse complement sequences) are listed in Additional file 21.
Recombinant protein expression and purification
Irga6 protein was expressed as N-terminal GST fusions from pGEX-4T-2 constructs in Escherichia coli BL21 upon overnight induction with 0.1 mM IPTG at 18°C. The cells were lysed in PBS (137 mM NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4)/2 mM DTT/Complete Mini Protease Inhibitor Cocktail EDTA free (Roche, Grenzach-Wyhlen, Germany) using a microfluidiser (EmulsiFlex-C5; Avestin, Ottawa, Ontario, Canada). Cleared lysates were purified on a GSTrap FF glutathione Sepharose affinity column (GE Healthcare, Munich, Germany) in PBS/2 mM DTT. GST was cleaved off by overnight incubation of the resin with thrombin (Serva, Heidelberg, Germany) at 4°C. Irga6 was eluted with PBS/2 mM DTT. Protein containing fractions were subjected to size exclusion chromatography (Superdex 75; GE Healthcare, Munich, Germany) in B1 buffer (50 mM Tris/HCl pH 7.4, 5 mM MgCl2)/2 mM DTT. Irga6 containing fractions were concentrated with Vivaspin 20 centrifugal concentrator (Sartorius, Goettingen, Germany). When indicated, an abbreviated protein purification procedure was used; omitting size exclusion chromatography and purifying Irga6 by glutathione affinity chromatography only.
Oligomerisation of Irga6 was monitored in B1 buffer/2 mM DTT by conventional or dynamic light scattering (DLS). For both, conventional and DLS, the protein buffer solution (90 μl) was cleared by ultracentrifugation (100,000 g, 30 minutes, 4°C). The reaction was started by addition of ice cold nucleotide (10 μl) to the protein buffer solution. The reaction was mixed by pipetting and transferred immediately to a cuvette. Conventional light scattering was performed at 350 or 600 nm at 37°C in an Aminco-Bowman 2 Luminescence Spectrometer (SLM Instruments, Urbana, IL, USA) or a DM45 Spectrofluorimeter (Olis, Bogart, GA, USA). Due to the unit-less readout the values obtained from the two instruments cannot be directly compared. DLS was performed at 650 nm at 20°C or 37°C with a DynaPro-E-20-660 molecular sizing instrument (Protein Solutions; Wyatt Technologies, Santa Barbara, CA, USA). Data were obtained and analysed using the DYNAMICS 5 and 6 software. Values of hydrodynamic radius given on the ordinates reflect the population mean particle size. Note that the derived hydrodynamic radius is not equal to the real size of the oligomer. WT and mutant Irga6 (with the exception of D164R and D164K) were stable and did not aggregate at 37°C in the presence of GDP.
Nucleotide hydrolysis assays
Nucleotide hydrolysis was measured in B1 buffer/2 mM DTT either by thin layer chromatography (TLC) and autoradiography or by high performance liquid chromatography (HPLC). For TLC and autoradiography, Irga6 was incubated with the indicated amounts of unlabeled nucleotide and traces of radioactively labeled nucleotide. The reaction was separated on PEI Cellulose F TLC plates (Merck, Darmstadt, Germany) in 1 M acetic acid/0.8 M LiCl. Signals were detected with the BAS 1000 phosphoimager analysis system (Fujifilm, Duesseldorf, Germany) and quantified with AIDA Image Analyser 3 (Raytest, Straubenhardt, Germany) or ImageQuant TL 7 (GE Healthcare, Munich, Germany) software. For HPLC, the reaction was stopped by 10-fold dilution in 10 mM NaOH; nucleotides were separated by ion exchange chromatography (MiniQ 4.6/50 PE; GE Healthcare, Munich, Germany) in 10 mM NaOH over a NaCl gradient. Absorption at 254 nm was monitored. Unicorn 4.12 (GE Healthcare, Munich, Germany) was used for quantification of peak areas.
Nucleotide binding measurement
Nucleotide-binding affinities were determined by equilibrium titration of Irga6 in the range of 0 to 100 μM against 0.5 mM mant nucleotide in B1 buffer/2 mM DTT at 20°C. The mant nucleotide was excited at 355 nm, and monitored at 448 nm in an Aminco-Bowman 2 Luminescence Spectrometer (SLM Instruments, Urbana, IL, USA). Equilibrium dissociation constants were obtained as described by . SigmaPlot 9 (Systat, Chicago, IL, USA) was used for dissociation constant (Kd) calculation.
IFNγ-induced (200 U/ml) gs3T3 cells were lysed for one hour at 4°C in PBS/0.1% Thesit (Sigma-Aldrich, St.Louis, MO, USA)/3 mM MgCl2 /Complete Mini Protease Inhibitor Cocktail EDTA free (Roche, Grenzach-Wyhlen, Germany). Postnuclear supernatants were incubated at 4°C overnight with glutathione Sepharose 4B (GE Healthcare, Munich, Germany)-bound recombinant GST-Irga6 with 0.5 mM of the indicated nucleotide. Cellular proteins were eluted from the washed beads with 100 mM Tris/HCl pH 8.5/0.5% SDS for 30 minutes at room temperature and subjected to SDS-PAGE and Western blot. Irgb6 and Irgm3 were detected using mouse monoclonal antibodies B34  and anti-IGTP (BD Biosciences, Franklin Lakes, NJ, USA) respectively. Input of recombinant GST-Irga6 was monitored by Ponceau S staining.
Cell culture and T. gondiiinfection
T. gondii ME49 tachyzoites were passaged and used for infection of Irga6-deficient mouse embryonic fibroblasts (MEFs)  as described earlier . MEFs were transiently transfected with pGW1H-Irga6-cTag1  constructs using FuGENE 6 (Roche, Grenzach-Wyhlen, Germany) and stimulated with 200 U/ml IFNγ (Peprotech, Hamburg, Germany) for 24 hours followed by infection with T. gondii at a multiplicity of infection of 7 for 2 hours, synchronised by centrifugation. Cells were fixed in 3% paraformaldehyde for 15 minutes and used for indirect immunostaining.
Immunocytochemistry was performed as described earlier  using anti-cTag1 rabbit sera , anti-GRA7 mouse monoclonal antibodies [56, 57] and Alexa 488/555 labeled donkey anti-rabbit and anti-mouse sera (Molecular Probes, Darmstadt, Germany). Probes were analysed microscopically as described earlier . Intracellular parasites were identified by the staining pattern of the T. gondii protein GRA7.
GTP (Carl Roth, Karlsruhe, Germany and Sigma-Aldrich, St.Louis, MO, USA); GDP (Sigma-Aldrich, St.Louis, MO, USA); GTPγS, XTP, 2'deoxy-GTP, mant-GTP, mant-GDP, mant-GTPγS, 2'mant-3'deoxy-GTP, 2'deoxy-3'mant-GTP, mant-XTP and mant-XDP (Jena Bioscience, Jena, Germany); 3'deoxy-GTP (Jena Bioscience, Jena, Germany and Trilink Biotechnologies, San Diego, CA, USA); 2'3'dideoxy-GTP (GE Healthcare, Munich, Germany); α32P-GTP (GE Healthcare, Munich, Germany, Hartmann Analytic, Braunschweig, Germany and Perkin Elmer, Waltham, MA, USA); γ32P-3'dGTP (Hartmann Analytic, Braunschweig, Germany)
Swiss-PdbViewer  was used for construction of structural models. CNSsolve  module buried surface  was used for calculation of contact surfaces. ClustalW2  was used for protein sequence alignment generation. ConSurf [62, 63] was used for calculation of conservation. PyMOL 0.99 (DeLano Scientific, Palo Alto, CA, USA) was used for image generation.
dynamic light scattering
GTPase activating protein
GDP dissociation inhibitor
high performance liquid chromatography
mouse embryonic fibroblast
parasitophorous vacuole membrane
signal recognition particle receptor
signal recognition particle
thin layer chromatography
We would like to thank Robert Finking for assistance in preparation of recombinant Irga6 protein. We thank Tobias Steinfeldt and Lan Tong for providing data before publication. We thank Gerrit J. Praefcke for help in determination of nucleotide binding affinities and valuable discussions. We are grateful to Thomas Langer and Reinhard Krämer for use of their laboratory equipment. AK and JPH were supported in part by the International Graduate School for Genetics and Functional Genomics and by the Graduate School for Biological Sciences of the University of Cologne. EW was supported from the Deutsche Forschungsgemeinschaft by Heisenberg-Stipendium grant Wo-695/4. This work was supported in part by the following grants to JCH from the Deutsche Forschungsgemeinschaft: SPP1110, Innate Immunity; SFB670, Cell Autonomous Immunity; SFB635 Post-translational Control of Protein Function; SFB680 Molecular Basis of Evolutionary Innovation.
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