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- activated human gene. Specific binding of hGBP1 to guanine nucleotides has been established although only two classical GTP-binding motifs were found in its primary sequence. The unique position of hGBP1 amongst known GTPases is further demonstrated by the hydrolysis of GTP to GDP and GMP. Although subsequent cleavage of orthophosphates rather than pyrophosphate was demonstrated, GDP coming from bulk solution cannot serve as a substrate. The relation of guanine nucleotide binding and hydrolysis to the antiviral function of hGBP1 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. Furthermore, we identified the third canonical GTP-binding motif and verified its role in nucleotide recognition by mutational analysis. The high guanine nucleotide dissociation rates measured by stopped-flow kinetics are responsible for the weak affinities to hGBP1 when compared to other GTPases like Ras or Galpha. By means of fluorescence and NMR spectroscopy it is demonstrated that aluminium fluoride forms a complex with hGBP1 only in the GDP state, presumably mimicking the transition state of GTP hydrolysis. Tentatively, 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 differential biological functions of the three nucleotide states and for the elucidation of the unique mechanism of nucleotide hydrolysis catalysed by hGBP1.
Praefcke
G J
GJ
Abteilung Strukturelle Biologie, Max-Planck-Institut für Molekulare Physiologie, Dortmund, 44202, Germany.
Geyer
M
M
Schwemmle
M
M
Robert Kalbitzer
H
H
Herrmann
C
C
D017607:Aluminum Compounds / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D002151:Calorimetry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D005459:Fluorides / Q000737:chemistry; D005470:Fluorometry; D019204:GTP-Binding Proteins; D006153:Guanosine Diphosphate / Q000378:metabolism; D006157:Guanosine Monophosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry; D013816:Thermodynamics
D017607:Aluminum Compounds; D004268:DNA-Binding Proteins; C490287:GBP1 protein, human; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D006157:Guanosine Monophosphate; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D005459:Fluorides; C032311:aluminum fluoride