REFINED X-RAY STRUCTURE OF DICTYOSTELIUM-DISCOIDEUM NUCLEOSIDE DIPHOSPHATE KINASE AT 1.8 ANGSTROM RESOLUTION

The X-ray structure of the nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum has been refined at 1.8 Angstrom resolut ion from a hexagonal crystal form with a 17 kDa monomer in its asymmet ric 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 0.188 and the estimated accuracy of the average atomic position, 0.25 Angstr om. The Dictyostelium structure is described in detail and compared to those of Drosophila and Myxcoccus xanthus NDP kinases. The protein is a hexamer with D3 symmetry. Residues 8 to 138 of each subunit form a globular alpha/beta domain. The four-stranded beta-sheet is antiparall el; its topology is different from other phosphate transfer enzymes, a nd also from the HPr protein which, like NDP kinase, carries a phospho rylated histidine. The same topology is nevertheless found in several other proteins that bind mononucleotides, RNA or DNA. Strand connectio ns in NDP kinase involve alpha-helices and a 20-residue segment called the Kpn loop. The beta-sheet is regular except for a beta-bulge in ed ge strand beta(2) and a gamma-turn at residue Ile120 just preceding st rand beta(4). The latter mag induce strain in the main chain near the active site His122. The alpha(1) beta(2) mot if participates in formin g dimers within the hexamer, helices alpha(1) and alpha(3) the Kpn loo p and C terminus, in forming trimers. The subunit fold and dimer inter actions found in Dictyostelium are conserved in other NDP kinases. Tri mer interactions probably occur in all eukaryotic enzymes. They are ab sent in the bacterial Myxococcus xanthus enzyme which is a tetramer, e ven though the subunit structure is very similar. In Dictyostelium, co ntacts between Kpn loops near the 3-fold axis block access to a centra l cavity lined with polar residues and filled with well-defined solven t molecules. Biochemical data on point mutants highlight the contribut ion of the Kpn loop to protein stability. In Myxococcus, the Kpn loops are on the tetramer surface and their sequence is poorly conserved. Y et, their conformation is maintained and they make a similar contribut ion to the substrate binding site.