The GTPase activity of murine guanylate-binding protein 2 (mGBP2) controls the intracellular localization and recruitment to the parasitophorous vacuole of Toxoplasma gondii.
One of the most abundantly IFN-γ-induced protein families in different cell types is the 65-kDa guanylate-binding protein family that is recruited to the parasitophorous vacuole of the intracellular parasite Toxoplasma gondii. Here, we elucidate the relationship between biochemistry and cellular host defense functions of mGBP2 in response to Toxoplasma gondii. The wild type protein exhibits low affinities to guanine nucleotides, self-assembles upon 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) using a Homo-FRET assay 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.
Institute of Medical Microbiology and Hospital Hygiene, Heinrich-Heine University, D-40225 Dusseldorf, Germany.
Elisabeth
E
Kravets
Daniel
D
Degrandi
Stefanie
S
Weidtkamp-Peters
Britta
B
Ries
Carolin
C
Konermann
Suren
S
Felekyan
Julia M
JM
Dargazanli
Gerrit J K
GJ
Praefcke
Claus A M
CA
Seidel
Lutz
L
Schmitt
Sander H J
SH
Smits
Klaus
K
Pfeffer
D000818:Animals; D019204:GTP-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006157:Guanosine Monophosphate / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007113:Immunity, Innate; D007371:Interferon-gamma / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D014122:Toxoplasma; D014123:Toxoplasmosis / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D014617:Vacuoles / Q000201:enzymology* / Q000235:genetics / Q000276:immunology / Q000469:parasitology
D006153:Guanosine Diphosphate; D007371:Interferon-gamma; D006157:Guanosine Monophosphate; D019204:GTP-Binding Proteins; C452200:Gbp2 protein, mouse