title: Role of individual domains and identification of internal gap in human guanylate binding protein-1.
abstract: Unlike other GTPases, interferon-gamma-induced human guanylate binding protein-1 has the ability to hydrolyze GTP to both GDP and GMP, with GMP being the major product of the reaction. This protein has two domains, an N-terminal globular domain and a C-terminal helical domain. These two domains are connected by a short intermediate region consisting of a two-stranded beta-sheet and a helix. As human guanylate binding protein-1 has been shown to undergo stimulated GTPase activity without external GTPase-activating protein, we sought to understand the roles of each of the two individual domains, the intermediate region, a conserved motif ((103)DXEKGD(108)), and the mechanism of the stimulation of GTPase activity. The steady-state assays using radiolabeled [alpha-(32)P]GTP on the wild-type protein suggest that the stimulation of activity primarily occurs during the cleavage of the second phosphate of GTP rather than the first, through allosteric interaction. Using several truncated and mutant proteins, we demonstrate for the first time that both the alpha-helix of the intermediate region and the (103)DXEKGD(108) motif play critical roles for the hydrolysis to GMP, but they appear to act in different ways: alpha-helix acts through structural stabilization by allosteric interaction and, thus, acts as an internal GTPase-activating protein, whereas the motif might act by providing necessary catalytic residues. Our data also show that the N-terminal globular domain is able to perform only the first catalysis (GTP to GDP, an activity associated with basal level), but the helical domain in the full-length protein stimulates the hydrolysis of GTP to GMP with higher GMP formation by preventing the dissociation of GDP-bound enzyme dimer.
authors.affiliation: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
authors.forename: Nazish
authors.initials: N
authors.lastname: Abdullah
authors.forename: Bharani
authors.initials: B
authors.lastname: Srinivasan
authors.forename: Nir
authors.initials: N
authors.lastname: Modiano
authors.forename: Peter
authors.initials: P
authors.lastname: Cresswell
authors.forename: Apurba Kumar
authors.initials: AK
authors.lastname: Sau
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D020134:Catalytic Domain; D002942:Circular Dichroism; D019281:Dimerization; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion
chemical_list: C490287:GBP1 protein, human; D050505:Mutant Proteins; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins