J Biol Chem. 2012 Jun 22. [Epub ahead of print]
The GTPase activity of murine guanylate-binding protein 2 (mGBP2) controls the intracellular localization and recruitment to the parasitophorous vacuole of Toxoplasma gondii
Kravets E, Degrandi D, Weidtkamp-Peters S, Ries B, Konermann C, Felekyan S, Dargazanli JM, Praefcke GJ, Seidel CA, Schmitt L, Smits SH, Pfeffer K.
Heinrich-Heine University, Germany;
One of the most abundantly IFN-γ-induced protein families in different cell types are the 65-kDa guanylate-binding proteins (GBPs) which are recruited to the parasitophorous vacuole (PV) of the intracellular parasite T. gondii. Here, we elucidate the relationship between biochemistry and cellular host defense functions of mGBP2 in response to T. gondii. The wild type (WT) protein exhibits low affinities to guanine nucleotides, self-assembles upon guanosine 5' triphosphate (GTP) binding, forming tetramers in the activated state and stimulates the GTPase activity in a cooperative manner. The products of the two consecutive hydrolysis reactions are both GDP and GMP. The biochemical characterization of point mutants in the GTP-binding motifs of mGBP2 revealed amino acid residues that decrease the GTPase activity by orders of magnitude and strongly impair nucleotide binding and multimerization ability. Live cell imaging employing Multiparameter Fluorescence Image Spectroscopy (MFIS) shows that the inducible multimerization of mGBP2 is dependent on a functional GTPase-domain. The consistent results indicate that GTP-binding, self-assembly and stimulated hydrolysis activity are required for physiological localization of the protein in infected and uninfected cells. Ultimately, we show that the GTPase-domain regulates efficient recruitment to T. gondii in response to IFN-γ.
PMID: 22730319 [PubMed - as supplied by publisher]