The activation mechanism of Irga6, an interferon-inducible GTPase contributing to mouse resistance against Toxoplasma gondii

Abstract

BackgroundThe 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.ResultsIrga6 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.ConclusionsThe 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.

Toxoplasma GRA7 effector increases turnover of immunity-related GTPases and contributes to acute virulence in the mouse

“…GRA7 was unable to bind to mutants (T102 and T108) that fail to hydrolyze GTP, and hence do not undergo oligomerization (16). Similarly, GRA7 was only able to bind to Irga6 in the presence of 2′dGTP, which induces polymerization more slowly than GTP, whereas it did not bind to Irga6 in the presence of 3′dGTP, which fails to drive oligomerization (27).…”

“…GRA7 coprecipitated recombinant Irga6 in the presence of GTP but not GDP (Fig. 4C), suggesting that it recognizes GTP-bound dimers and/or oligomers but not GDP-bound monomers of Irga6 (26,27). Precipitation was not due to formation of large oligomers or aggregates of Irga6 and GRA7, as shown by the failure of these proteins to pellet in the absence of Ni beads (Fig.…”

“…Intriguingly, GRA7 only bound to forms of Irga6 that were competent for oligomerization. Structural and modeling studies have defined a dimer interface in Irga6 that is necessary for homo-oligomerization and mediates binding to heterodimer formation with IRGM proteins that negatively regulate polymer assembly (27). GRA7 was unable to bind to mutants (T102 and T108) that fail to hydrolyze GTP, and hence do not undergo oligomerization (16).…”

“…To test the specificity of the GRA7-Irga6 interaction further, we took advantage of previous studies that have defined the importance of GTP hydrolysis on the formation of Irga6 oligomers. Irga6 binds to 2′dGTP and 3′ dGTP but only oligomerizes with 2′dGTP (27). GRA7 bound to Irga6 in the presence of 2′dGTP but not 3′dGTP (Fig.…”

The Toxoplasma Pseudokinase ROP5 Is an Allosteric Inhibitor of the Immunity-related GTPases

“…IRG Oligomerization-Oligomerization of IRGa6 was measured by an increase in turbidity at A 400 and was carried out essentially as described previously (30). Briefly, oligomerization of 60 M IRGa6 alone or in the presence of 100 M wild-type or mutant ROP5C I was initiated by the addition of 5 mM GTP.…”