STRUCTURAL STUDIES OF NUCLEOSIDE DIPHOSPHATE KINASE

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The broad long-term objective of the proposed work is to determine the structure and understand the catalytic mechanism of nucleoside diphosphate (NDF) kinase, an enzyme that plays a key role in nucleotide metabolism. Exciting new research is beginning to shed light on the enzyme's ability to suppress metastasis as well as its ability to regulate the composition of nucleotide pools. Structural analyses of these proteins will provide a foundation for modulating the cellular functions incumbent upon this enzyme as well as providing a basis for improving the anti-neoplastic and anti-viral properties of some nucleotide analogs. We have recently determined the 2.0 Angstrom resolution X-ray crystallographic structure of the Myxococcus xanthus enzyme. We have a crystal form of the enzyme that is enzymatically active. With these crystals, we have been able to collect 1.7 Angstrom resolution data for complexes of the enzyme with nucleotides. The structures of the complexes reveal that the enzyme has a novel nucleotide binding motif that is quite different than two predictions for the mode of nucleotide binding-one based upon the sequence analysis and one based upon the structure of an inactive mutant of the Dictyostelium discoideum NDF kinase. The NDP kinases function via a ping-pong mechanism in which an autophosphorylated enzyme intermediate is formed. The intermediate of the enzymatic mechanism is a phosphohistidine. The structure suggests a mechanism for this phosphorylation. Though many enzymes form phosphohistidine intermediates, no structure has been determined for any of them. We have been able to stabilize the NDP kinase phosphoenzyme intermediate sufficiently for X-ray crystallographic data collection. Based on structural information, we are examining the roles of active-site mutants by site-directed mutagenesis and steady-state enzyme kinetics. We will determine the three-dimensional structures of NDP kinase complexed with each of 12 different substrates and inhibitors. These studies of NDP kinase-substrate complexes will provide a structural basis for rational design of more effective nucleotide analogs of anti-neoplastic and anti- viral importance. Human and murine NDP kinase Nm23 is a suppressor of metastasis in some cell types. We have obtained crystals of a human NDP kinase, Nm23-H2. In addition to its enzymatic activity, Nm23-H2 is a DNA-binding protein that is a specific transcriptional activator of the c-myc oncogene in vitro. These crystals diffract to a resolution limit of 3.0 Angstrom. We will determine the structure of the human enzyme with molecular replacement techniques using our structure of the M. xanthus enzyme. We will also determine the structure of a complex of Nm23-H2 with a double-stranded oligonucleotide.