Complex Formation and the GTP Hydrolysis Mechanism of the Immunity-Related GTPase Irga6

Immunity-related GTPases mediate resistance against a broad spectrum of intracellular pathogens. Irga6 (IIGP1) accumulates on the membrane of the parasitophorous vacuole, which harbours the protozoan parasite Toxoplasma gondii inside of infected cells. The protein participates in disruption of the parasites domicile, and of the enclosed pathogen, via vesiculation and membrane stripping. The enzymatic properties of Irga6, low nucleotide-binding affinity, GTP-dependent oligomerisation and cooperative GTP hydrolysis, are to be found in the dynamin superfamily of large GTPases. Dynamin oligomerises at the neck of nascent vesicles, and mediates their scission from the plasma membrane. This study focuses on the Irga6 complex formation process, and attempts to explain how the catalytic activity is stimulated by the interaction of Irga6 molecules. One of the contact surfaces, engaged in Irga6 complex formation, the so-called catalytic-interface, which is a part of the G-domain and comprises the nucleotide-binding site, was defined. The molecular surface, provided by the bound nucleotide, was shown to be employed in Irga6 complex formation. A hypothetical model of the dimeric Irga6 topology, based on the unique substrate constellation and the catalytic machinery, found in the dimeric complex of the signal recognition particle and its receptor, was proposed. The crucial catalytic reciprocal 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 was shown to be essential for Irga6 complex formation, and activation of GTP hydrolysis in trans. The model further suggested a glutamate as a key catalytic residue, which activates the GTP hydrolysis. This glutamate was shown to be crucial for, complex formation mediated stimulation of, the enzymatic activity. The fundamental biochemical properties, of two further members of the protein family, Irgb6 and Irgd, were characterized. A preliminary analysis of Irgm3 demonstrated the, elsewhere predicted, inhibitory function of this protein on GTP hydrolysis by Irga6 and Irgb6

The immunity-related GTPase Irga6 dimerizes in a parallel head-to-head fashion

The immunity-related GTPases (IRGs) constitute a powerful cell-autonomous resistance system against several intracellular pathogens. Irga6 is a dynamin-like protein that oligomerizes at the parasitophorous vacuolar membrane (PVM) of Toxoplasma gondii leading to its vesiculation. Based on a previous biochemical analysis, it has been proposed that the GTPase domains of Irga6 dimerize in an antiparallel fashion during oligomerization.Leibniz Graduate School grants: (SFB958, SFB635)

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

Background: 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. Results: 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 (SRalpha). 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. Conclusions: 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

Regulatory interactions between IRG resistance GTPases in the cellular response to Toxoplasma gondii

Members of the immunity-related GTPase (IRG) family are interferon-inducible resistance factors against a broad spectrum of intracellular pathogens including Toxoplasma gondii. The molecular mechanisms governing the function and regulation of the IRG resistance system are largely unknown. We find that IRG proteins function in a system of direct, nucleotide-dependent regulatory interactions between family members. After interferon induction but before infection, the three members of the GMS subfamily of IRG proteins, Irgm1, Irgm2 and Irgm3, which possess an atypical nucleotide-binding site, regulate the intracellular positioning of the conventional GKS subfamily members, Irga6 and Irgb6. Following infection, the normal accumulation of Irga6 protein at the parasitophorous vacuole membrane (PVM) is nucleotide dependent and also depends on the presence of all three GMS proteins. We present evidence that an essential role of the GMS proteins in this response is control of the nucleotide-bound state of the GKS proteins, preventing their GTP-dependent activation before infection. Accumulation of IRG proteins at the PVM has previously been shown to be associated with a block in pathogen replication: our results relate for the first time the enzymatic properties of IRG proteins to their role in pathogen resistance

Phosphorylation of Mouse Immunity-Related GTPase (IRG) Resistance Proteins Is an Evasion Strategy for Virulent Toxoplasma gondii

GTPases of the mouse IRG protein family, mediators of resistance against Toxoplasma gondii in the mouse, are inactivated by a polymorphic kinase of the parasite, resulting in enhanced parasite virulence

Dynamin self-assembly and the vesicle scission mechanism How dynamin oligomers cleave the membrane neck of clathrin-coated pits during endocytosis

Recently, Gao et al. and Chappie et al. elucidated the crystal structures of the polytetrameric stalk domain of the dynamin-like virus resistance protein, MxA, and of the G-domain dimer of the large, membrane-deforming GTPase, dynamin, respectively. Combined, they provide a hypothetical oligomeric structure for the complete dynamin protein. Here, it is discussed how the oligomers are expected to form and how they participate in dynamin mediated vesicle fission during the process of endocytosis. The proposed oligomeric structure is compared with the novel mechanochemical model of dynamin function recently proposed by Bashkirov et al. and Pucadyil and Schmid. In conclusion, the new model of the dynamin oligomer has the potential to explain how short self-limiting fissogenic dynamin assemblies are formed and how concerted GTP hydrolysis is achieved. The oligomerisation of two other dynamin superfamily proteins, the guanylate binding proteins (GBPs) and the immunity-related GTPases (IRGs), is addressed briefly

Additional file 1: of The immunity-related GTPase Irga6 dimerizes in a parallel head-to-head fashion

Supplementary Material (all files combined). Table S1. Data collection statistics. Table S2. Refinement statistics. Figure S1. Mutations R31E, K32E, K176E, K246E eliminate GTP-dependent oligomerization and GTP hydrolysis of Irga6. Figure S2. Packing of Irga6 molecules in the crystal lattice. Figure S3. Contact surfaces in Irga6. Figure S4. Electron density map of the catalytic site in molecule A. (PDF 3.88 mb