Interferons are immunomodulatory cytokines that mediate anti-pathogenic and anti-proliferative effects in cells1. Interferon-γ-inducible human guanylate binding protein 1 (hGBP1) belongs to the family of dynamin-related large GTP-binding proteins2, which share biochemical properties not found in other families of GTP-binding proteins such as nucleotide-dependent oligomerization and fast cooperative GTPase activity3. hGBP1 has an additional property by which it hydrolyses GTP to GMP in two consecutive cleavage reactions4,5. Here we show that the isolated amino-terminal G domain of hGBP1 retains the main enzymatic properties of the full-length protein and can cleave GDP directly. Crystal structures of the N-terminal G domain trapped at successive steps along the reaction pathway and biochemical data reveal the molecular basis for nucleotide-dependent homodimerization and cleavage of GTP. Similar to effector binding in other GTP-binding proteins, homodimerization is regulated by structural changes in the switch regions. Homodimerization generates a conformation in which an arginine finger and a serine are oriented for efficient catalysis. Positioning of the substrate for the second hydrolysis step is achieved by a change in nucleotide conformation at the ribose that keeps the guanine base interactions intact and positions the β-phosphates in the γ-phosphate-binding site.
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We thank the ESRF for providing synchrotron radiation facilities and the staff of beamlines BM30A and ID14-EH1/4 for technical assistance during data collection; and E. Wolf for discussions, and M.-F. Carlier and J. Cherfils for support. This work was supported by grants from the Association pour la Recherche contre le Cancer (to L.R.) and the Boehringer Ingelheim Fonds (to G.J.K.P.).
Coordinates and structure factors have been deposited in the Protein Data Bank under accession numbers 2BC9 (GppNHp-bound hGBP1LG), 2B92 (GDP•AlF3-bound hGBP1LG), 2B8W (GMP•AlF4--bound hGBPLG) and 2D4H (GMP-bound hGBP1LG). Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.
The file contains Supplementary Methods, Supplementary Table 1 (nucleotide binding and GTP hydrolysis data), Supplementary Table 2 (nucleotide-dependent oligomerization states for both full-length hGBP1 (hGBP1FL) and LG domain of hGBP1 (hGBP1LG), Supplementary Table 3 (torsion angles of the ribose moiety in hGBP1, Ras and dynamin structures) and Supplementary Figure Legends. (DOC 180 kb)
Nucleotide binding and nucleotide-dependent oligomerization experiments of hGBP1LG. (PDF 10145 kb)
Sequence alignment of LG domains of human GBP homologues with residues involved in the hGBP1LG dimer interface and important mutated residues indicated. (PDF 118 kb)
Schematic view of the interactions for the active site of hGBP1LG in complexes with GppNHp•Mg2+, GDP•AlF3•Mg2+, GMP•AlF4-•Mg2+ and GMP. (PDF 7963 kb)
Conformational changes of switch 1, 2 and guanine cap regions between dimeric hGBP1LG•GMP•AlF4- and monomeric hGBP1LG•GMP crystal structures. (PDF 7205 kb)
Schematic drawing of the consecutive events during GTP binding, GTPase-, GDPase reaction, product release and dissociation, with the corresponding structural changes as discussed in the text. (PDF 3749 kb)
Views of electron density simulated-annealing omit maps around the nucleotide binding site for hGBP1LG•GppNHp, hGBP1LG•GDP•AlF3, hGBP1LG•GMP•AlF4- and hGBP1LG•GMP crystal structures. (PDF 8395 kb)
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Exposing catalytic versatility of GTPases: taking reaction detours in mutants of hGBP1 enzyme without additional energetic cost
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