BACKGROUND
immunity-related gtpases  are major contributors to cell autonomous resistance against the intracellular protozoal pathogen, toxoplasma gondii  <cit> .
for nomenclature of irg proteins, see methods and  <cit> .
multiple members of the family are expressed in cells induced by interferon-γ .
many irg proteins, including irga <dig>  and irgb <dig>  relocate from resting cytoplasmic compartments to the parasitophorous vacuole membrane  of avirulent t. gondii  <cit> .
loading of irg proteins onto the t. gondii pvm is followed by vesiculation and rupture of the pvm and death of the parasite  <cit> .
irga <dig> at the pvm is in the active, gtp-bound state, while cytoplasmic irga <dig> is inactive and probably gdp-bound  <cit> .
irga <dig> forms gtp-dependent oligomeric complexes in vitro and in vivo and hydrolysis of the gtp substrate is cooperatively activated  <cit> .
these enzymatic properties of irga <dig> together with the relatively high molecular mass of  <dig> kda and the nucleotide binding affinities in the micromolar range  <cit>  are also found in several other families of large gtpases, including members of the dynamin superfamily  <cit> , associated with membrane remodelling and, like mx proteins, resistance against intracellular pathogens  <cit> .
the structure of the irga <dig> protein was determined some years ago  <cit> .
the protein consists of a ras-like g-domain  <cit>  and a helical domain .
the g-domain contains three conserved gtp-binding motifs   <cit>  and two flexible switch regions, switch i and switch ii  <cit> .
homology considerations suggest that the structure of irga <dig> can provide a reasonable template for the irg family.
three members of the irg family, irgm <dig> , irgm <dig>  and irgm <dig> , carry a unique substitution of the otherwise universally conserved p-loop  lysine  to methionine    <cit> .
in the absence of gdp-dependent negative regulatory interactions with the three gms proteins, gks subfamily members including irga <dig> activate prematurely in the cytoplasm, form gtp-dependent aggregates, and are unable to accumulate on the pvm of invading t. gondii  <cit> .
little is known about the relationship between the gtp-dependent activation of irga <dig> 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 irga <dig> 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  and its receptor   <cit> .
we demonstrate that the catalytic interface includes the bound gtp substrate and that the 3'hydroxyl  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 irga <dig> with irgb <dig>  <cit>  and the inhibitory interaction between irga <dig> and the gms subfamily protein, irgm <dig>  <cit> .
lastly, we show that the integrity of the catalytic interface of irga <dig> is required for the accumulation of the active, gtp-bound protein at the t. gondii pvm.
RESULTS
the catalytic interface is localised on the g-domain
to gain insight into the mechanism of gtp-dependent oligomerisation and activation of irga <dig>  a number of solvent-exposed residues identified from the known crystal structure  <cit>  were mutated , in large part with bulky charged residues with the intention of disrupting putative intermolecular interactions.
mutations of a contiguous cluster of g-domain residues  essentially abolished oligomerisation  and gtp hydrolysis .
mutations of two further residues in this cluster, thr <dig> and thr <dig>  have been shown elsewhere to lose both functions  <cit> .
these residues define a surface , called the catalytic interface, that includes the conformationally labile switch i region , suggesting that the conformation of switch i is critical for dimer formation and activation.
mutations of the residues arg <dig>  lys <dig>  lys <dig>  lys <dig>  arg <dig> and lys <dig>  also reduced gtp-dependent oligomerisation to some extent , but none completely .
these residues formed a loosely defined "secondary patch" on the irga <dig> surface .
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 .
at present, therefore, we do not consider the secondary patch to be an oligomerisation interface.
the oligomerisation of irga <dig> was not prevented by numerous other mutations , suggesting the absence of a second well-defined surface interface contributing to oligomerisation.
the majority of catalytic interface mutants including t102a and t108a  <cit>  had no significant effect on the binding affinity for gtp .
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  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 .
furthermore, none of the mutants of the secondary patch, which all showed reduced nucleotide-binding affinities , prevented oligomerisation of irga <dig>
irga <dig> crystallizes as a rotationally symmetrical dimer  <cit>  .
mutants of the crystal dimer interface were seen to oligomerise less efficiently than the wild-type  and it was suggested that this interface might participate in cooperative gtp-dependent activation  <cit> .
the crystal dimer interface does not obstruct the catalytic interface described here  and could therefore contribute to active irga <dig> oligomerisation.
mutants of the four crystal dimer interface residues leu <dig>  lys <dig>  ser <dig> and met <dig>  that had been examined earlier  <cit> , were therefore re-assayed .
mutants of nine further residues  in the crystal dimer interface were also analysed .
under the conditions of these experiments, which were more stringent than those used previously, out of  <dig> residues mutated in the crystal dimer interface only the mutations of lys <dig> and lys <dig>  two residues already identified in the secondary patch, partially inhibited oligomerisation .
furthermore, the formation of the crystal dimer is not nucleotide-dependent  <cit> , whereas the oligomerisation of irga <dig> requires gtp binding  <cit> .
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  <cit>  of irga <dig> , 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 irga <dig> dimer
although usually discussed in the context of dynamin and its relatives, irga <dig> also resembles in several respects two gtpases of the srp family, ffh  and ftsy .
ffh, ftsy and irga <dig> share a wide-open nucleotide-binding pocket in their nucleotide-bound state  <cit> .
ffh  <cit> , ftsy  <cit>  and irga <dig>  <cit>  share nearly identical low nucleotide-binding affinities, caused by a high substrate dissociation rate.
ffh and ftsy are significantly homologous to each other and form a gtp-dependent heterodimer.
each acts in trans as a gtpase activating protein  for the other in the dimeric complex  <cit> .
irga <dig> molecules work as mutual gaps in gtp-dependent oligomeric complexes  <cit> .
the common biochemical and structural properties suggested that the mechanism of cooperative hydrolysis known from ffh and ftsy could be relevant for understanding the catalytic mechanism of irga <dig>  the key to this idea is the catalytic interaction between the two closely opposed gtp molecules in anti-parallel orientation and, specifically, the crystallographically determined, catalytically important reciprocal trans interactions between the 3'ohs and the γ-phosphates  <cit> .
coordinates of the nucleotides from the crystal structure of the ffh-ftsy heterodimer from egea and colleagues   <cit>   were used to define the position in space of the nucleotides bound by two irga <dig> molecules .
the irga6-m173a gppnhp complex structure  was used for this analysis since this structure was the only one that resolved all residues between glu <dig> and the c-terminal asn <dig>   <cit> .
in the irga <dig> dimer model thus constructed  the two irga <dig> molecules complement each other well, apart from one overlap of the two atomic structures made by the side chain of arg <dig> .
the buried surface area in the hypothetical irga <dig> dimer is  <dig> Å <dig> .
the modeled catalytic interface surface is in good agreement with the mutagenesis data.
all the residues where mutation destroyed oligomerisation are located within or proximal to the contact area of the dimer subunits .
the only exception is lys <dig>  localised just inside the margin of the catalytic interface surface though oriented outwards , where mutation had no effect on oligomerisation .
the properties of lys <dig> and arg <dig> may indicate the occurrence of conformational changes that, by analogy with ffh-ftsy  <cit> , are expected to accompany gtp binding and complex formation.
the ribose of the bound nucleotide is part of the catalytic interface
at the core of the irga <dig> dimer model the 2' and 3'ohs of gtp ribose form part of the contact surface.
modifications of the nucleotide ribose at the 2' or 3'oh would therefore be expected to interfere with oligomerisation.
oligomerisation of irga <dig> in the presence of 2'/3'o--gtp  was investigated.
mant is a small fluorescent group bound via the 2' or 3'oxygen to the gtp ribose in mant-gtp, a nucleotide analog used in affinity determinations .
consistent with the lack of free space between the subunits of the dimer model, mant-gtp was unable to stimulate oligomerisation of irga <dig> .
mant-group-dependent inhibition of complex formation was also observed for irga <dig> protein immunoprecipitated from cells .
the inhibitory effect of the mant-group is not caused by reduced nucleotide binding  <cit> , implying as predicted from the dimer model that the gtp ribose is part of the interaction interface between irga <dig> oligomer subunits.
the base of bound nucleotide is part of the catalytic interface
in the irga <dig> dimer model the bound nucleotides are part of the interaction interface of the two subunits.
the specificity of gtpases for guanine nucleotides is determined by a conserved aspartate in the g4-motif.
this aspartate interacts with the exocyclic amino-group of the guanine ring at the c <dig> position .
the substitution of the g <dig> aspartate by asparagine changes the nucleotide specificity of gtpases from guanine to xanthine nucleotides, which have an oxo-group at the c <dig> position .
the d251n mutation in the g4-motif of ffh changes the binding preference of the protein from gtp to xanthosine-5'triphosphate .
it was shown that gtp-initiated complex formation between the two srp gtpases, ffh-d251n and ftsy, is inhibited by addition of xtp  <cit> .
the c <dig> amino-group is part of the interaction surface between ffh and ftsy therefore the binding of xtp to ffh-d251n alters the interface and inhibits complex formation.
the nucleotide-binding preference of irga <dig> was changed from guanine to xanthine based nucleotides by the corresponding g4-motif mutation d186n .
unexpectedly, despite a nine-fold higher affinity for xtp than for gtp, irga6-d186n hydrolysed gtp more efficiently than xtp .
oligomerisation of irga6-d186n  accompanied by gtpase activity  could be activated by gtp, albeit inefficiently, and both were abolished when the high affinity ligand, xtp, was added at a concentration 1/ <dig> that of gtp .
this shows that the replacement of the surface exposed c <dig> amino-group, of bound gtp, by the oxo-group, of xtp, inhibits irga <dig> 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 c <dig> amino-group are glu <dig> and ser <dig> .
consistently, mutations of glu <dig> and ser <dig> both caused loss of oligomerisation .
the 3'oh of the gtp ribose is required for trans-activation of gtp hydrolysis
the model of the irga <dig> dimer is based on reciprocal trans interactions between the ribose 3'ohs and the γ-phosphates of the opposed nucleotides  <cit> , analogous to those shown to be crucial for the reciprocal activation of gtp hydrolysis between the paired gtpases, ffh and ftsy  <cit> .
the involvement of the 2' and 3'ohs of gtp in irga <dig> oligomerisation and gtp hydrolysis was investigated.
oligomerisation of irga <dig> could be stimulated by gtp and 2'deoxy-gtp , both of which have the 3'oh.
in contrast, no complex formation was observed in presence of 3'deoxy-gtp  and 2'3'dideoxy-gtp , both of which lack the 3'oh .
consistent with these results, only basal hydrolysis rates of about  <dig>  min- <dig> were found for 3'dgtp and 2'3'ddgtp in contrast to efficient hydrolysis of gtp or 2'dgtp .
the oligomerisation rate in presence of 2'dgtp was somewhat reduced , consistent with the idea that the 2'oh is part of the catalytic interface, as suggested by the model.
but, unlike the 3'oh the 2'oh is not required for cooperative hydrolysis .
the removal of the 2' or 3'oh of the gtp ribose decreased the nucleotide-binding affinity slightly .
however, as already argued, the kd variation alone cannot be responsible for the observed inability of 3'dgtp and 2'3'ddgtp to stimulate irga <dig> oligomerisation at millimolar nucleotide concentrations.
for the ffh-ftsy heterodimer the essential activation function of the 3'oh is mediated in trans  <cit> .
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 irga <dig> 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 irga <dig> 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 .
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.
glu <dig> is a key residue crucial for the activation of catalysis
the core of the irga <dig> dimer model is created by the reciprocal trans interactions of the gtp 3'ohs with the γ-phosphates.
these enforce a specific relative orientation of the two nucleotides and, therefore, also of the two protein molecules to which the nucleotides are bound.
the dimer model suggests additional trans interactions between the 3'ohs and the glu <dig> residues .
the involvement of glu <dig> in the activation of gtp hydrolysis was investigated.
glu <dig> is part of the catalytic interface .
the mutants e106k and e106r essentially abolished oligomerisation .
however some exchanges like e106d, e106q, e106a and e106n, could oligomerise to a considerable extent .
nevertheless, no mutation of glu <dig> could activate hydrolysis, whether oligomerising or not .
thus, glu <dig> is a residue essential for the activation of gtp hydrolysis independently of a role in the contact interface.
glu <dig> is part of the flexible switch i region which undergoes nucleotide-dependent conformational changes  <cit> .
in the gdp state glu <dig> 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 .
however, in the gppnhp state glu <dig> can be reoriented towards the γ-phosphate of the bound nucleotide  <cit> .
the gtp ribose 3'oh may stabilize the glu <dig> residue in trans in a conformation allowing complex formation and in an orientation required for activation of the catalytic water molecule in cis .
this could initiate a nucleophilic attack on the γ-phosphate and activate gtp hydrolysis.
the catalytic interface of irga <dig> is essential for heteromeric interactions between irg members
in addition to forming gtp-dependent homomeric complexes in vitro  <cit>  and in vivo  <cit>  irga <dig> can also form heteromeric nucleotide-dependent complexes with other members of the irg family.
the gks protein, irgb <dig>  interacts strongly with irga <dig> in yeast 2-hybrid assays in a nucleotide-dependent manner  <cit> .
this is a positive interaction that assists the accumulation of irga <dig> on the pvm of t. gondii  <cit> .
we could observe this interaction in a pull-down assay between recombinant glutathione s-transferase -tagged irga <dig> and irgb <dig> from ifnγ-induced cell lysates .
the interaction was gtpγs-dependent and failed in the presence of gdp .
the importance of the catalytic interface for this interaction was demonstrated by the complete failure of gtpγs-dependent pull-downs with catalytic interface mutants of irga <dig> .
the three gms proteins, irgm <dig>  irgm <dig> and irgm <dig>  are essential negative regulators of irga <dig>  <cit> .
for irgm <dig> this interaction has been shown to be gdp-dependent and inhibited by gtpγs  <cit> .
in cells, in the absence of this interaction, irga <dig> binds gtp, activates spontaneously, and cannot accumulate on pvms of invading t. gondii  <cit> .
we were able to confirm the previously documented gdp-dependent interaction between ifnγ-induced irgm <dig> and recombinant irga <dig> in a pull-down assay, and additionally showed that no interaction occurred when mant-gdp was used , 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 irgm <dig> with irga <dig> .
the two residues whose mutation did not interfere with irga6-irgm <dig> interaction, gly <dig> and ser <dig>  are located in a different part of the catalytic interface from arg <dig> and asn <dig>  these results suggest that the gdp-dependent negative regulatory interaction between irgm <dig> and irga <dig> 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 irga <dig> to the t. gondii pvm
in ifnγ stimulated cells about 60% of irga <dig> colocalises with the endoplasmic reticulum  while the remainder is cytosolic  <cit> .
upon infection with t. gondii the protein accumulates on the pvm  <cit> , depleting other cytoplasmic compartments.
irg protein-dependent destruction of the pvm and subsequent death of the parasite occurs in ifnγ-induced cells  <cit> .
it is not known whether the process of irga <dig> oligomer formation is causally connected with immunity against t. gondii.
this issue was addressed in the context of the accumulation of irga <dig> at the t. gondii pvm  <cit> .
irga6-deficient cells, stimulated with ifnγ and transiently transfected with wt or mutant irga <dig>  were infected with the avirulent t. gondii strain me <dig> .
wt irga <dig> accumulated on the pvm, while all mutants of the catalytic interface showed quantitatively  and qualitatively  drastically reduced recruitment to the pvm.
the sole exception was lys <dig>  located at the rim of the catalytic interface and the secondary patch .
mutations of this residue inhibited oligomerisation and catalytic activity in vitro  but did not prevent accumulation of the mutant protein on the pvm .
the contribution of lys <dig> to the catalytic interface is ambiguous: the structural model places the residue just inside the interface.
it is possible that the configuration of the irga <dig> catalytic interface in vivo differs slightly from that in vitro.
this could be due to a conformational effect of the myristoyl group, which is exposed by gtp binding in vivo  <cit>  and required for vacuolar accumulation .
equally, the predominant catalytic dimers that form in vivo may be heterodimers between irga <dig> and other irg proteins rather than irga <dig> homodimers  <cit> .
heterodimeric catalytic interfaces may be slightly different from the irga <dig> homodimer interface assayed here.
discussion
irga <dig> forms gtp-dependent oligomers and gtp hydrolysis is activated in this state  <cit> .
the present study has identified a new catalytic interface  required for the formation of irga <dig> oligomers.
this interface provides a platform for both positive and negative nucleotide-dependent regulatory interactions between irga <dig> molecules and other members of the irg protein family .
these interactions are essential for the activity of the irg proteins in resistance to t. gondii   <cit> .
the revealed surface is part of the g-domain, including the nucleotide-binding site and the switch regions .
the nucleotide itself is part of the interface .
structural and biochemical features common to the srp gtpases and irga <dig> suggested a model  for the irga <dig> dimer based on the relative orientation of the two nucleotides buried in the srp-srα complex  <cit> .
the mutagenesis data were consistent with the proposed model , 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  <cit> .
in strong support for the validity of the srp-srα based model of the irga <dig> dimer, the 3'oh of the gtp ribose proved to be absolutely required for oligomerisation and gtp hydrolysis by irga <dig> and, as in srp-srα, this function was exercised in trans only .
functionally, irga <dig> seems closer to the dynamins in that it is involved in the vesiculation and disruption of the pvm  <cit> , 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  and belong, in fact, to the two different major clades  that have been defined over multiple gtpase families  <cit> .
if indeed, irg proteins share the unusual catalytic mechanism of srp  <cit>  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 irga <dig> 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  <cit> .
in contrast, the srp-srα paired gtpases engage the two monomers in an anti-parallel orientation with the two nucleotides in reciprocal atomic contact  <cit> .
we explored the feasibility of alternative models of the irga <dig> 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 .
none provided a satisfactory basis on which to explain the properties of irga <dig>  in models based on ehd <dig>  <cit> , meab  <cit>  and mnme  <cit> , the two irga <dig> g-domains interact via different surfaces that do not include the bound nucleotides.
the models based on sept <dig>  <cit> , gimap <dig>  <cit> , bdlp  <cit> , toc <dig>  <cit>  and soj  <cit>  engage small parts of the catalytic interface in limited interfaces.
the catalytic interface is involved in the models based on hypb  <cit>  and av <dig>  <cit> .
the model based on hgbp <dig>  <cit>  involves the catalytic interface but the subunits overlap in the contact area.
the dynamin  <cit>  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 irga <dig> 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 .
the activation of gtp hydrolysis in irga <dig> complexes
gaps work by supplementation of missing catalytic residues , and by reorientation and stabilization of the catalytic machinery which is already present in the target protein  <cit> .
the model of the irga <dig> dimer suggests that the switch regions are stabilized by the interaction of the two irga <dig> molecules.
in particular, the model suggests that glu <dig>  is stabilized by the trans interaction with the 3'oh of the gtp ribose .
mutational analysis of glu <dig>  together with structural data  <cit>  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  is consistent with the anticipated function of glu <dig>
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  <cit> .
in contrast to the irga <dig> 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  <cit> .
it may be relevant that acidic residues have been implicated in activating the catalytic water in further dimer-forming gtpases, meab  <cit> , mnme  <cit>  and hypb  <cit> , as also in related atpases, soj  <cit>  and av <dig>  <cit> .
the irga <dig> dimer model does not suggest any positively-charged residue that could fulfill an arginine finger-like function.
the mutation of the most promising candidate, lys <dig> in switch i, to glutamate had no effect on complex formation or gtp hydrolysis .
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  <cit> .
these interactions recall the proposed trans interactions of the 3'oh of the gtp ribose with the γ-phosphate and glu <dig> in the irga <dig> dimer model .
generally, the transition state in irga <dig> 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  <cit> , 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 irga <dig> surface .
it probably represents a central platform engaged in functional interactions between irg proteins in general.
heteromeric interactions between irga <dig> and other members of the irg family play important regulatory roles in the biological action of irga <dig>  while irgb <dig> enhances the accumulation of activated irga <dig>  <cit>  on the t. gondii pvm  <cit> , irgm <dig> prevents the premature activation of irga <dig> prior to infection by locking the gdp-bound state of the protein  <cit> .
in vitro, the catalytic interface is involved in the gtp-dependent irga6-irgb <dig> interaction  and also in the gdp-dependent irga6-irgm <dig> interaction .
these results show that the negative regulatory interaction between irga <dig> and irgm <dig> occurs, like the activating irga6-irga <dig> and irga6-irgb <dig> interactions, via the catalytic interface.
the outcome of the irga6-irgm <dig> interaction thus resembles the primary action of a gdp dissociation inhibitor   <cit> .
thus two different functions  seem to be mediated through the catalytic interface.
all tested mutants of the catalytic interface prevented the irga6-irgb <dig> interaction , but the irga6-irgm <dig> interaction was prevented only by a subset of the mutants  suggesting a distinct mode of interaction.
the catalytic interface of the gms proteins, including irgm <dig>  contains specific substitutions .
the otherwise conserved residues glu <dig>  asp <dig> and arg <dig>  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 irga <dig> have deleterious effects on gtp-dependent complex formation and hydrolysis activation .
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. gondii virulence factor
upon infection with avirulent t. gondii irga <dig> accumulates at the pvm and participates in disruption of the pvm and killing of the parasite  <cit> .
the accumulation of irg proteins at the pvm is a prerequisite for the antimicrobial function  <cit> .
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 irga <dig> on the pvm of avirulent t. gondii .
irga <dig>  with other irg proteins, does not accumulate normally on the pvm of virulent t. gondii and the parasites survive and continue to replicate  <cit> .
the secreted rop <dig> kinase  <cit>  is a major virulence factor of t. gondii  <cit> .
the significance of the catalytic interface for the function of irga <dig> is highlighted by the recent finding that the virulence-associated rop <dig> kinase from virulent, but not avirulent, t. gondii strains phosphorylates conserved threonine residues, thr <dig> and thr <dig>  in switch i within the catalytic interface of irga <dig>  thus blocking oligomerisation, gtpase activity and the accumulation of irga <dig> at the pvm  <cit> .
CONCLUSIONS
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 , for defense against t. gondii.
the irg protein, irga <dig>  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 irga <dig> is activated in oligomeric complexes formed by the protein.
we define one of the contact surfaces involved in irga <dig> 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 irga <dig> with irgb <dig> and irgm <dig> 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 irga <dig> 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 irga <dig>  based on the unique substrate geometry and catalytic machinery found in the dimeric complex of the signal recognition particle, ffh , and its receptor, ftsy .
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 irga <dig> 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 irga <dig>  we consider that the functional similarity between these two gtpase families is probably the result of convergent evolution.

 immunity-related gtpases are major contributors to cell autonomous resistance against the intracellular protozoal pathogen, toxoplasma gondii. for nomenclature of irg proteins, see methods and. multiple members of the family are expressed in cells induced by interferon-γ. many irg proteins, including irga and irgb relocate from resting cytoplasmic compartments to the parasitophorous vacuole membrane of avirulent t. gondii. loading of irg proteins onto the t. gondii pvm is followed by vesiculation and rupture of the pvm and death of the parasite. irga at the pvm is in the active, gtp-bound state, while cytoplasmic irga is inactive and probably gdp-bound. irga forms gtp-dependent oligomeric complexes in vitro and in vivo and hydrolysis of the gtp substrate is cooperatively activated. these enzymatic properties of irga together with the relatively high molecular mass of 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. the structure of the irga protein was determined some years ago. the protein consists of a ras-like g-domain and a helical domain. the g-domain contains three conserved gtp-binding motifs and two flexible switch regions, switch i and switch ii. homology considerations suggest that the structure of irga can provide a reasonable template for the irg family. three members of the irg family, irgm, irgm and irgm, carry a unique substitution of the otherwise universally conserved p-loop lysine to methionine. in the absence of gdp-dependent negative regulatory interactions with the three gms proteins, gks subfamily members including irga activate prematurely in the cytoplasm, form gtp-dependent aggregates, and are unable to accumulate on the pvm of invading t. gondii. little is known about the relationship between the gtp-dependent activation of irga 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 irga 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 and its receptor. we demonstrate that the catalytic interface includes the bound gtp substrate and that the 3'hydroxyl 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 irga with irgb and the inhibitory interaction between irga and the gms subfamily protein, irgm. lastly, we show that the integrity of the catalytic interface of irga 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 to gain insight into the mechanism of gtp-dependent oligomerisation and activation of irga a number of solvent-exposed residues identified from the known crystal structure were mutated, in large part with bulky charged residues with the intention of disrupting putative intermolecular interactions. mutations of a contiguous cluster of g-domain residues essentially abolished oligomerisation and gtp hydrolysis. mutations of two further residues in this cluster, thr and thr have been shown elsewhere to lose both functions. these residues define a surface, called the catalytic interface, that includes the conformationally labile switch i region, suggesting that the conformation of switch i is critical for dimer formation and activation. mutations of the residues arg lys lys lys arg and lys also reduced gtp-dependent oligomerisation to some extent, but none completely. these residues formed a loosely defined "secondary patch" on the irga surface. 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. at present, therefore, we do not consider the secondary patch to be an oligomerisation interface. the oligomerisation of irga was not prevented by numerous other mutations, 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. 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 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. furthermore, none of the mutants of the secondary patch, which all showed reduced nucleotide-binding affinities, prevented oligomerisation of irga irga crystallizes as a rotationally symmetrical dimer. mutants of the crystal dimer interface were seen to oligomerise less efficiently than the wild-type 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 and could therefore contribute to active irga oligomerisation. mutants of the four crystal dimer interface residues leu lys ser and met that had been examined earlier, were therefore re-assayed. mutants of nine further residues in the crystal dimer interface were also analysed. under the conditions of these experiments, which were more stringent than those used previously, out of residues mutated in the crystal dimer interface only the mutations of lys and lys two residues already identified in the secondary patch, partially inhibited oligomerisation. furthermore, the formation of the crystal dimer is not nucleotide-dependent, whereas the oligomerisation of irga 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 irga, 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 irga dimer although usually discussed in the context of dynamin and its relatives, irga also resembles in several respects two gtpases of the srp family, ffh and ftsy. ffh, ftsy and irga share a wide-open nucleotide-binding pocket in their nucleotide-bound state. ffh, ftsy and irga share nearly identical low nucleotide-binding affinities, caused by a high substrate dissociation rate. ffh and ftsy are significantly homologous to each other and form a gtp-dependent heterodimer. each acts in trans as a gtpase activating protein for the other in the dimeric complex. irga molecules work as mutual gaps in gtp-dependent oligomeric complexes. the common biochemical and structural properties suggested that the mechanism of cooperative hydrolysis known from ffh and ftsy could be relevant for understanding the catalytic mechanism of irga the key to this idea is the catalytic interaction between the two closely opposed gtp molecules in anti-parallel orientation and, specifically, the crystallographically determined, catalytically important reciprocal trans interactions between the 3'ohs and the γ-phosphates. coordinates of the nucleotides from the crystal structure of the ffh-ftsy heterodimer from egea and colleagues were used to define the position in space of the nucleotides bound by two irga molecules. the irga6-m173a gppnhp complex structure was used for this analysis since this structure was the only one that resolved all residues between glu and the c-terminal asn. in the irga dimer model thus constructed the two irga molecules complement each other well, apart from one overlap of the two atomic structures made by the side chain of arg. the buried surface area in the hypothetical irga dimer is Å. the modeled catalytic interface surface is in good agreement with the mutagenesis data. all the residues where mutation destroyed oligomerisation are located within or proximal to the contact area of the dimer subunits. the only exception is lys localised just inside the margin of the catalytic interface surface though oriented outwards, where mutation had no effect on oligomerisation. the properties of lys and arg may indicate the occurrence of conformational changes that, by analogy with ffh-ftsy, are expected to accompany gtp binding and complex formation. the ribose of the bound nucleotide is part of the catalytic interface at the core of the irga dimer model the 2' and 3'ohs of gtp ribose form part of the contact surface. modifications of the nucleotide ribose at the 2' or 3'oh would therefore be expected to interfere with oligomerisation. oligomerisation of irga in the presence of 2'/3'o--gtp was investigated. mant is a small fluorescent group bound via the 2' or 3'oxygen to the gtp ribose in mant-gtp, a nucleotide analog used in affinity determinations. consistent with the lack of free space between the subunits of the dimer model, mant-gtp was unable to stimulate oligomerisation of irga. mant-group-dependent inhibition of complex formation was also observed for irga protein immunoprecipitated from cells. the inhibitory effect of the mant-group is not caused by reduced nucleotide binding, implying as predicted from the dimer model that the gtp ribose is part of the interaction interface between irga oligomer subunits. the base of bound nucleotide is part of the catalytic interface in the irga dimer model the bound nucleotides are part of the interaction interface of the two subunits. the specificity of gtpases for guanine nucleotides is determined by a conserved aspartate in the g4-motif. this aspartate interacts with the exocyclic amino-group of the guanine ring at the c position. the substitution of the g aspartate by asparagine changes the nucleotide specificity of gtpases from guanine to xanthine nucleotides, which have an oxo-group at the c position. the d251n mutation in the g4-motif of ffh changes the binding preference of the protein from gtp to xanthosine-5'triphosphate. it was shown that gtp-initiated complex formation between the two srp gtpases, ffh-d251n and ftsy, is inhibited by addition of xtp. the c amino-group is part of the interaction surface between ffh and ftsy therefore the binding of xtp to ffh-d251n alters the interface and inhibits complex formation. the nucleotide-binding preference of irga was changed from guanine to xanthine based nucleotides by the corresponding g4-motif mutation d186n. unexpectedly, despite a nine-fold higher affinity for xtp than for gtp, irga6-d186n hydrolysed gtp more efficiently than xtp. oligomerisation of irga6-d186n accompanied by gtpase activity could be activated by gtp, albeit inefficiently, and both were abolished when the high affinity ligand, xtp, was added at a concentration 1/ that of gtp. this shows that the replacement of the surface exposed c amino-group, of bound gtp, by the oxo-group, of xtp, inhibits irga 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 c amino-group are glu and ser. consistently, mutations of glu and ser both caused loss of oligomerisation. the 3'oh of the gtp ribose is required for trans-activation of gtp hydrolysis the model of the irga dimer is based on reciprocal trans interactions between the ribose 3'ohs and the γ-phosphates of the opposed nucleotides, analogous to those shown to be crucial for the reciprocal activation of gtp hydrolysis between the paired gtpases, ffh and ftsy. the involvement of the 2' and 3'ohs of gtp in irga oligomerisation and gtp hydrolysis was investigated. oligomerisation of irga could be stimulated by gtp and 2'deoxy-gtp, both of which have the 3'oh. in contrast, no complex formation was observed in presence of 3'deoxy-gtp and 2'3'dideoxy-gtp, both of which lack the 3'oh. consistent with these results, only basal hydrolysis rates of about min- were found for 3'dgtp and 2'3'ddgtp in contrast to efficient hydrolysis of gtp or 2'dgtp. the oligomerisation rate in presence of 2'dgtp was somewhat reduced, consistent with the idea that the 2'oh is part of the catalytic interface, as suggested by the model. but, unlike the 3'oh the 2'oh is not required for cooperative hydrolysis. the removal of the 2' or 3'oh of the gtp ribose decreased the nucleotide-binding affinity slightly. however, as already argued, the kd variation alone cannot be responsible for the observed inability of 3'dgtp and 2'3'ddgtp to stimulate irga oligomerisation at millimolar nucleotide concentrations. 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 irga 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 irga 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. 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. glu is a key residue crucial for the activation of catalysis the core of the irga dimer model is created by the reciprocal trans interactions of the gtp 3'ohs with the γ-phosphates. these enforce a specific relative orientation of the two nucleotides and, therefore, also of the two protein molecules to which the nucleotides are bound. the dimer model suggests additional trans interactions between the 3'ohs and the glu residues. the involvement of glu in the activation of gtp hydrolysis was investigated. glu is part of the catalytic interface. the mutants e106k and e106r essentially abolished oligomerisation. however some exchanges like e106d, e106q, e106a and e106n, could oligomerise to a considerable extent. nevertheless, no mutation of glu could activate hydrolysis, whether oligomerising or not. thus, glu is a residue essential for the activation of gtp hydrolysis independently of a role in the contact interface. glu is part of the flexible switch i region which undergoes nucleotide-dependent conformational changes. in the gdp state glu 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. however, in the gppnhp state glu can be reoriented towards the γ-phosphate of the bound nucleotide. the gtp ribose 3'oh may stabilize the glu residue in trans in a conformation allowing complex formation and in an orientation required for activation of the catalytic water molecule in cis. this could initiate a nucleophilic attack on the γ-phosphate and activate gtp hydrolysis. the catalytic interface of irga is essential for heteromeric interactions between irg members in addition to forming gtp-dependent homomeric complexes in vitro and in vivo irga can also form heteromeric nucleotide-dependent complexes with other members of the irg family. the gks protein, irgb interacts strongly with irga in yeast 2-hybrid assays in a nucleotide-dependent manner. this is a positive interaction that assists the accumulation of irga on the pvm of t. gondii. we could observe this interaction in a pull-down assay between recombinant glutathione s-transferase -tagged irga and irgb from ifnγ-induced cell lysates. the interaction was gtpγs-dependent and failed in the presence of gdp. the importance of the catalytic interface for this interaction was demonstrated by the complete failure of gtpγs-dependent pull-downs with catalytic interface mutants of irga. the three gms proteins, irgm irgm and irgm are essential negative regulators of irga. for irgm this interaction has been shown to be gdp-dependent and inhibited by gtpγs. in cells, in the absence of this interaction, irga binds gtp, activates spontaneously, and cannot accumulate on pvms of invading t. gondii. we were able to confirm the previously documented gdp-dependent interaction between ifnγ-induced irgm and recombinant irga in a pull-down assay, and additionally showed that no interaction occurred when mant-gdp was used, 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 irgm with irga. the two residues whose mutation did not interfere with irga6-irgm interaction, gly and ser are located in a different part of the catalytic interface from arg and asn these results suggest that the gdp-dependent negative regulatory interaction between irgm and irga 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 irga to the t. gondii pvm in ifnγ stimulated cells about 60% of irga colocalises with the endoplasmic reticulum while the remainder is cytosolic. upon infection with t. gondii the protein accumulates on the pvm, depleting other cytoplasmic compartments. irg protein-dependent destruction of the pvm and subsequent death of the parasite occurs in ifnγ-induced cells. it is not known whether the process of irga oligomer formation is causally connected with immunity against t. gondii. this issue was addressed in the context of the accumulation of irga at the t. gondii pvm. irga6-deficient cells, stimulated with ifnγ and transiently transfected with wt or mutant irga were infected with the avirulent t. gondii strain me. wt irga accumulated on the pvm, while all mutants of the catalytic interface showed quantitatively and qualitatively drastically reduced recruitment to the pvm. the sole exception was lys located at the rim of the catalytic interface and the secondary patch. mutations of this residue inhibited oligomerisation and catalytic activity in vitro but did not prevent accumulation of the mutant protein on the pvm. the contribution of lys to the catalytic interface is ambiguous: the structural model places the residue just inside the interface. it is possible that the configuration of the irga catalytic interface in vivo differs slightly from that in vitro. this could be due to a conformational effect of the myristoyl group, which is exposed by gtp binding in vivo and required for vacuolar accumulation. equally, the predominant catalytic dimers that form in vivo may be heterodimers between irga and other irg proteins rather than irga homodimers. heterodimeric catalytic interfaces may be slightly different from the irga homodimer interface assayed here. discussion irga forms gtp-dependent oligomers and gtp hydrolysis is activated in this state. the present study has identified a new catalytic interface required for the formation of irga oligomers. this interface provides a platform for both positive and negative nucleotide-dependent regulatory interactions between irga molecules and other members of the irg protein family. these interactions are essential for the activity of the irg proteins in resistance to t. gondii. the revealed surface is part of the g-domain, including the nucleotide-binding site and the switch regions. the nucleotide itself is part of the interface. structural and biochemical features common to the srp gtpases and irga suggested a model for the irga dimer based on the relative orientation of the two nucleotides buried in the srp-srα complex. the mutagenesis data were consistent with the proposed model, 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. in strong support for the validity of the srp-srα based model of the irga dimer, the 3'oh of the gtp ribose proved to be absolutely required for oligomerisation and gtp hydrolysis by irga and, as in srp-srα, this function was exercised in trans only. functionally, irga 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 and belong, in fact, to the two different major clades that have been defined over multiple gtpase families. if indeed, irg proteins share the unusual catalytic mechanism of srp 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 irga 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. in contrast, the srp-srα paired gtpases engage the two monomers in an anti-parallel orientation with the two nucleotides in reciprocal atomic contact. we explored the feasibility of alternative models of the irga 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. none provided a satisfactory basis on which to explain the properties of irga in models based on ehd, meab and mnme, the two irga g-domains interact via different surfaces that do not include the bound nucleotides. the models based on sept, gimap, bdlp, toc and soj engage small parts of the catalytic interface in limited interfaces. the catalytic interface is involved in the models based on hypb and av. the model based on hgbp 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 irga 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. the activation of gtp hydrolysis in irga complexes gaps work by supplementation of missing catalytic residues, and by reorientation and stabilization of the catalytic machinery which is already present in the target protein. the model of the irga dimer suggests that the switch regions are stabilized by the interaction of the two irga molecules. in particular, the model suggests that glu is stabilized by the trans interaction with the 3'oh of the gtp ribose. mutational analysis of glu 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 is consistent with the anticipated function of glu 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. in contrast to the irga 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. 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 av. the irga dimer model does not suggest any positively-charged residue that could fulfill an arginine finger-like function. the mutation of the most promising candidate, lys in switch i, to glutamate had no effect on complex formation or gtp hydrolysis. 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. these interactions recall the proposed trans interactions of the 3'oh of the gtp ribose with the γ-phosphate and glu in the irga dimer model. generally, the transition state in irga 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, 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 irga surface. it probably represents a central platform engaged in functional interactions between irg proteins in general. heteromeric interactions between irga and other members of the irg family play important regulatory roles in the biological action of irga while irgb enhances the accumulation of activated irga on the t. gondii pvm, irgm prevents the premature activation of irga prior to infection by locking the gdp-bound state of the protein. in vitro, the catalytic interface is involved in the gtp-dependent irga6-irgb interaction and also in the gdp-dependent irga6-irgm interaction. these results show that the negative regulatory interaction between irga and irgm occurs, like the activating irga6-irga and irga6-irgb interactions, via the catalytic interface. the outcome of the irga6-irgm interaction thus resembles the primary action of a gdp dissociation inhibitor. thus two different functions seem to be mediated through the catalytic interface. all tested mutants of the catalytic interface prevented the irga6-irgb interaction, but the irga6-irgm interaction was prevented only by a subset of the mutants suggesting a distinct mode of interaction. the catalytic interface of the gms proteins, including irgm contains specific substitutions. the otherwise conserved residues glu asp and arg 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 irga have deleterious effects on gtp-dependent complex formation and hydrolysis activation. 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. gondii virulence factor upon infection with avirulent t. gondii irga accumulates at the pvm and participates in disruption of the pvm and killing of the parasite. the accumulation of irg proteins at the pvm is a prerequisite for the antimicrobial function. 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 irga on the pvm of avirulent t. gondii. irga with other irg proteins, does not accumulate normally on the pvm of virulent t. gondii and the parasites survive and continue to replicate. the secreted rop kinase is a major virulence factor of t. gondii. the significance of the catalytic interface for the function of irga is highlighted by the recent finding that the virulence-associated rop kinase from virulent, but not avirulent, t. gondii strains phosphorylates conserved threonine residues, thr and thr in switch i within the catalytic interface of irga thus blocking oligomerisation, gtpase activity and the accumulation of irga 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, for defense against t. gondii. the irg protein, irga 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 irga is activated in oligomeric complexes formed by the protein. we define one of the contact surfaces involved in irga 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 irga with irgb and irgm 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 irga 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 irga based on the unique substrate geometry and catalytic machinery found in the dimeric complex of the signal recognition particle, ffh, and its receptor, ftsy. 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 irga 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 irga we consider that the functional similarity between these two gtpase families is probably the result of convergent evolution. 
it therefore seems probable, if surprising, that the substrate-assisted catalytic mechanism has been independently evolved in the two protein families. 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. irga forms gtp-dependent oligomers in vitro and thereby activates hydrolysis of the gtp substrate. 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. 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 irga from accumulating on the parasitophorous vacuole membrane of t. gondii, showing that gtp-dependent irga activation is an essential component of the resistance mechanism. the catalytic interface of irga 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. 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. the irg protein, irga, participates in the disruption of the vacuolar membrane surrounding the intracellular parasite, toxoplasma gondii, through which it communicates with its cellular hosts. understanding the activation mechanism of irga will help to explain the mechanism by which irg proteins exercise their resistance function. in this study we define the catalytic g-domain interface by mutagenesis and present a structural model, of how gtp hydrolysis is activated in irga complexes, based on the substrate-twinning reaction mechanism of the signal recognition particle and its receptor.
 the interferon-inducible immunity-related gtpases are a distinctive family of gtpases that function as powerful cell-autonomous resistance factors. the irg protein, irga, 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.  irga 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 irga complexes, based on the substrate-twinning reaction mechanism of the signal recognition particle and its receptor. 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 irga from accumulating on the parasitophorous vacuole membrane of t. gondii, showing that gtp-dependent irga activation is an essential component of the resistance mechanism.  the catalytic interface of irga 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 irga 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. 