IIGP1, an interferon-gamma-inducible 47-kDa GTPase of the mouse, showing cooperative enzymatic activity and GTP-dependent multimerization.
IIGP1 belongs to a well defined family of 47-kDa GTPases whose members are present at low resting levels in mouse cells but are strongly induced transcriptionally by interferons and are implicated in cell-autonomous resistance to intracellular pathogens. Recombinant IIGP1 was expressed in Escherichia coli and purified to homogeneity. Here we present a detailed biochemical characterization of IIGP1 using various biochemical and biophysical methods. IIGP1 binds to GTP and GDP with dissociation constants in the micromolar range with at least 10 times higher affinity for GDP than for GTP. IIGP1 hydrolyzes GTP to GDP, and the GTPase activity is concentration-dependent with a GTP turnover rate of 2 min-1 under saturating protein concentrations. Functional interaction between IIGP1 molecules is shown by nucleotide-dependent oligomerization in vitro. Both cooperative hydrolysis of GTP and GTP-dependent oligomerization are blocked in a mutant form of IIGP1 modified at the C terminus. IIGP1 shares micromolar nucleotide affinities and oligomerization-dependent hydrolytic activity with the 67-kDa GTPase hGBP1 (induced by type I and type II interferons), with the antiviral Mx proteins (interferon type I-induced) and with the paradigm of the self-activating large GTPases, the dynamins, with which Mx proteins show homology. The higher relative affinity for GDP and the relatively low GTPase activity distinguish IIGP1, but this study clearly adds IIGP1 and thus the p47 GTPases to the small group of cooperative GTPase families that appear to characterize the development of intracellular resistance during the interferon response to infection. The present analysis provides essential parameters to understand the molecular mechanism by which IIGP1 participates in this complex resistance program.
Uthaiah
Revathy C
RC
Institute for Genetics, University of Cologne, Zülpicher Strasse 47, 50674 Cologne, Germany.
Praefcke
Gerrit J K
GJ
Howard
Jonathan C
JC
Herrmann
Christian
C
D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D006868:Hydrolysis; D007113:Immunity, Innate; D007239:Infections; D007700:Kinetics; D051379:Mice; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; D020558:GTP Phosphohydrolases; C491070:Iigp1 protein, mouse