Journal of Molecular Biology
Volume 243, Issue 5, 10 November 1994, Pages 873-890
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Regular Article
Refined X-ray Structure of Dictyostelium discoideum Nucleoside Diphosphate Kinase at 1.8 Å resolution

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Abstract

The X-ray structure of the nucleoside diphosphate kinase (NDP kinace) from Dictyostelium discoideum has been refined at 1.8 Å resolution from a hexagonal crystal form with a 17 kDa monomer in its asymmetric unit. The atomic model was derived from the previously determined structure of a point mutant of the protein. It contains 150 amino acid residues out of 155, and 95 solvent molecules. The R-factor is 0.196 and the estimated accuracy of the average atomic position, 0.25 Å. The Dictyotelium structure is described in detail and compared to those of Drosophila and Myxococcua xanthus NDP kinases. The protein is a hexamer with D3 symmetry. Residues 8 to 138 of each subunit form a globular α/β domain. The four-stranded β-sheet is antiparallel; its topology is different from other phosphate transfer enzymes, and also from the HPr protein which, like NDP kinase, carries a phosphorylated histidine. The same topology is nevertheless found in several other proteins that bind mononucleotides, RNA or DNA. Strand connections in NDP kinase involve α-helices and a 20-residue segment called the Kpn loop. The β-sheet is regular except for a β-bulge in edge strand β2 and a γ-turn at residue Ile120 just preceding strand β4. The latter may induce strain in the main chain near the active site His122. The α1β2 motif participates in forming dimers within the hexamer, helices α1, and α3, the Kpn loop and C terminus, in forming trimers. The subunit fold and dimer interactions found in Dictyostelium are conserved in other NDP kinases. Trimer interactions probably occur in all eukaryotic enzymes. They are absent in the bacterial Myxococus xanthus enzyme which is a tetramer, even though the subunit structure is very similar. In Dictyostelium, contacts between Kpn loops near the 3-fold axis block access to a central cavity lined with polar residues and filled with well-defined solvent molecules. Biochemical data on point mutants highlight the contribution of the Kpn loop to protein stability. In Myxococcus, the Kpn loops are on the tetramer surface and their sequence is poorly conserved. Yet, their conformation is maintained and they make a similar contribution to the substrate binding site.

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