Interferon-induced MxA protein. GTP binding and GTP hydrolysis properties.
MxA is a GTPase encoded by an interferon-activated human gene which inhibits the multiplication of several RNA viruses. Recombinant histidine-tagged MxA protein (His-MxA) was expressed in Escherichia coli and purified to near homogeneity. Gel filtration showed that it formed high molecular weight oligomers. Purified His-MxA exhibited specific GTP hydrolysis rates of up to 350 nmol of GTP/min/mg of protein, corresponding to a turnover number of 27 min-1. The Km for this reaction was 260 microM. Guanine nucleotides did not copurify with His-MxA. Binding experiments in solution with fluorescent-labeled nucleotides confirmed that His-MxA binds guanine nucleotides rather weakly and further showed that the fluorescent GDP analog N-methylanthraniloyl (mant)-GDP had a much lower affinity for His-MxA (Kd 20 microM, koff 8.5 s-1) than the nonhydrolyzable GTP analog mant-5'-guanylyl-beta,gamma-imidotriphosphate (mant-GMP-PNP) (Kd 0.75 microM, koff 0.012 s-1). Competitive binding studies with nonlabeled nucleotides revealed a similar binding preference of His-MxA for GTP over GDP: the Kd for GTP was 20 microM, whereas the Kd for GDP was 100 microM. Thus, a high percentage of MxA molecules may be complexed with GTP in vivo.
Richter
M F
MF
Abteilung Virologie, Universität Freiburg, Germany.
Schwemmle
M
M
Herrmann
C
C
Wittinghofer
A
A
Staeheli
P
P
D000998:Antiviral Agents / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001704:Biopolymers; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007372:Interferons / Q000502:physiology*; D007700:Kinetics; D064547:Myxovirus Resistance Proteins; D011485:Protein Binding; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D000998:Antiviral Agents; D001704:Biopolymers; C579071:MX1 protein, human; D064547:Myxovirus Resistance Proteins; D011506:Proteins; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D006639:Histidine; D006160:Guanosine Triphosphate; D007372:Interferons; D019204:GTP-Binding Proteins