Nucleotide-binding characteristics of human guanylate-binding protein 1 (hGBP1) and identification of the third GTP-binding motif

hGBP1 is a GTPase with antiviral activity encoded by an interferon-activate d human gene. Specific binding of hGBP1 to guanine nucleotides has been est ablished although only two classical GTP-binding motifs were found in its p rimary sequence. The unique position of hGBP1 amongst known GTPases is furt her demonstrated by the hydrolysis of Gm to GDP and GMP. Although subsequen t cleavage of orthophosphates rather than pyrophosphate was demonstrated, G DP coming from bulk solution cannot serve as a substrate. The relation of g uanine nucleotide binding and hydrolysis ito the antiviral function of hGBP 1 is unknown. Here we show similar binding affinities for all three guanine nucleotides and the ability of both products, GDP and GMP, to compete with GTP binding. Fluorimetry and isothermal titration calorimetry were applied to prove that only one nucleotide binding site is present in hGBP1. Furthe rmore, we identified the third canonical GTP-binding motif and verified its role in nucleotide recognition by mutational analysis. The :high guanine n ucleotide dissociation rates :measured by stopped-flow kinetics are respons ible for the weak affinities to hGBP1 when compared to other GTPases like P as or G(alpha). By means of fluorescence and NMR spectroscopy it is demonst rated that aluminium fluoride forms a complex with hGBP1 only in the GDP sl ate, presumably mimicking the transition state of GTP hydrolysis. Tentative ly, the involvement of a GAP domain in hGBP1 in GTP hydrolysis is suggested . These results will serve as a basis for the determination of the differen tial biological functions of the three nucleotide states and for the elucid ation of the unique mechanism of nucleotide hydrolysis catalysed by hGBP1. (C) 1999 Academic Press.