Abstract
The X-ray crystallographic structure of nucleoside diphosphate (NDP) kinase from
Myxococcus xanthus has been determined using multiple isomorphous replacement techniques and refined at 2·0 Å resolution to a crystallographic
R-factor of 0·17. This is the first report of the structure of an enzymatically active NDP kinase and of the enzyme with a bound nucleotide. The structure has been determined in
P 4
32
12 and
I222 crystal forms. The enzyme monomer consists of a four-stranded antiparallel βsheet. The surfaces of the sheet are partially covered with five helical segments. There are two protein molecules in the asymmetric unit of the tetragonal crystal form. They form a direct with an extensive interface in which 1092 Å
2 per monomer is buried. The majority, of the contact area in the dimer interface is between hydrophobic or aromatic residues. Two dimers are related by a crystallographic 2-fold axis to yield a tetramer. This tetramer is also present in the orthorhombic crystals; however, in this case, the 222 symmetry is entirely crystallographic. Upon tetramer formation, an additional 473 Å
2 of solvent-accessible surface area from each monomer becomes buried. The interface between dimers in the tetramer is stabilized by salt bridges. Equilibrium sedimentation studies are consistent with the enzyme being a tetramer in solution.
The structure of a complex of adenosine diphosphate (ADP) with the enzyme was determined and reveals that most of the nucleotide interactions with the protein are with the pyrophosphate and ribose groups, while the base has no hydrogen bonds with the protein and interacts only by stacking with the side chain of Phe59. The Mg
2+ interacts with the pyrophosphate of the ADP and
via a solvent molecule with the side chain of the conserved Asp120 residue. The mode of interaction with the nucleotide is novel, with the nucleotide binding at the side of the β-sheet.
The structures of the nucleotide in crystals grown in the presence or absence of Mg
2+ are essentially identical. In addition, the phosphotransfer reaction from adenosine triphosphate (ATP) to the enzyme can occur without Mg
2+. This suggests that only the second step of the reaction in which the enzyme transfers the phosphate to a nucleoside diphosphate acceptor is significantly catalyzed by the metal.