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 K-m for this reaction was 260 mu M. 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 (K-d 20 mu M, k(off) 8.5 s(-1)) than the nonhydrolyzable GTP analog mant-5'-guanylyl-beta,gamma-imidotriphosphate (mant-GMP-PNP) (K-d 0.75 mu M, k(off) 0.012 s(-1)). Competitive binding studies with nonlabeled nucleotides revealed a similar binding preference of His-MxA for GTP over GDP: the K-d for GTP was 20 mu M, whereas the K-d for GDP was 100 mu M. Thus, a high percentage of MxA molecules may be complexed with GTP in vivo.