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.
Highlights
The interferon-inducible immunity-related GTPases (IRG proteins/p47 GTPases) are a distinctive family of GTPases that function as powerful cell-autonomous resistance factors
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
The catalytic interface is localised on the G-domain To gain insight into the mechanism of GTP-dependent oligomerisation and activation of Irga6, a number of solvent-exposed residues identified from the known crystal structure [14] were mutated (Figure 1), in large part with bulky charged residues with the intention of disrupting putative intermolecular interactions
Summary
The interferon-inducible immunity-related GTPases (IRG proteins/p47 GTPases) are a distinctive family of GTPases that function as powerful cell-autonomous resistance factors. Irga 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 Irga together with the relatively high molecular mass of 47 kDa and the nucleotide binding affinities in the micromolar range [11] are found in several other families of large GTPases, including members of the dynamin superfamily [12], associated with membrane remodelling and, like Mx proteins, resistance against intracellular pathogens [1,13]. 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 [9,10,19]
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