title: The activation mechanism of Irga6, an interferon-inducible GTPase contributing to mouse resistance against Toxoplasma gondii.
abstract: 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 (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.
  
  
  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.
authors.affiliation: Institute for Genetics, Department of Cell Genetics, University of Cologne, Zülpicher Strasse 47a, 50674 Cologne, Germany.
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Pawlowski
authors.forename: Aliaksandr
authors.initials: A
authors.lastname: Khaminets
authors.forename: Julia P
authors.initials: JP
authors.lastname: Hunn
authors.forename: Natasa
authors.initials: N
authors.lastname: Papic
authors.forename: Andreas
authors.initials: A
authors.lastname: Schmidt
authors.forename: Revathy C
authors.initials: RC
authors.lastname: Uthaiah
authors.forename: Rita
authors.initials: R
authors.lastname: Lange
authors.forename: Gabriela
authors.initials: G
authors.lastname: Vopper
authors.forename: Sascha
authors.initials: S
authors.lastname: Martens
authors.forename: Eva
authors.initials: E
authors.lastname: Wolf
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Howard
mesh_terms: D000818:Animals; D020558:GTP Phosphohydrolases / Q000276:immunology* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006790:Host-Parasite Interactions; D007113:Immunity, Innate; D051379:Mice; D008810:Mice, Inbred C57BL; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D018000:Receptors, Peptide / Q000378:metabolism; D018271:Signal Recognition Particle / Q000378:metabolism; D014122:Toxoplasma / Q000378:metabolism*; D014617:Vacuoles / Q000276:immunology* / Q000378:metabolism / Q000469:parasitology
chemical_list: D018160:Receptors, Cytoplasmic and Nuclear; D018000:Receptors, Peptide; D018271:Signal Recognition Particle; C036318:signal peptide receptor; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; C491070:Iigp1 protein, mouse