Abstract
Nucleoside diphosphate (NDP) kinase catalyzes the phosphorylation of ribo- and deoxyribonucleosides diphosphates into triphosphates. NDP kinase is also involved in malignant tumors and was shown to activate in vitro transcription of the c-myc oncogene by binding to its NHE sequence. The structure of the complex of NDP kinase with bound ADP shows that the nucleotide adopts a different conformation from that observed in other phosphokinases with an internal H bond between the 3'-OH and the beta-O made free by the phosphate transfer. We use intrinsic protein fluorescence to investigate the inhibitory and binding potential of nucleotide analogues phosphorylated in 3'-OH position of the ribose to both wild type and F64W mutant NDP kinase from Dictyostelium discoideum. Due to their 3'-phosphate, 5'-phosphoadenosine 3'-phosphate (PAP) and adenosine 3'-phosphate 5'-phosphosulfate (PAPS) can be regarded as structural analogues of enzyme-bound ADP. The KD of PAPS (10 microM) is three times lower than the KD of ADP. PAPS also acts as a competitive inhibitor toward natural substrates during catalysis, with a KI in agreement with binding data. The crystal structure of the binary complex between Dictyostelium NDP kinase and PAPS was solved at 2.8-A resolution. It shows a new mode of nucleotide binding at the active site with the 3'-phosphate of PAPS located near the catalytic histidine, at the same position as the gamma-phosphate in the transition state. The sulfate group is directed toward the protein surface. PAPS will be useful for the design of high affinity drugs targeted to NDP kinases.
Highlights
Nucleoside diphosphate (NDP)1 kinase (EC 2.7.4.6) catalyzes the phosphorylation of nucleotides diphosphates into triphosphates using ATP as the major phosphate donor
The structure of the complex of NDP kinase with bound ADP shows that the nucleotide adopts a different conformation from that observed in other phosphokinases with an internal H bond between the 3-OH and the -O made free by the phosphate transfer
Crystallization and Structure Analysis of Binary Complexes—Binary complexes were obtained by soaking phosphate 5-phosphosulfate (PAPS) in crystals of Dictyostelium NDP kinase grown in hanging drops under the following conditions: 30 –32% polyethylene glycol 550, 50 mM Tris-HCl, pH 8.5, and 20 mM MgCl2 in the reservoir; 5 mg/ml protein, 15–16% polyethylene glycol 550, 50 mM Tris-HCl, pH 8.5, 20 mM MgCl2, and 17 mM 3Ј-amino-ADP in the drop
Summary
Materials—ATP, dTDP, cAMP, lactate dehydrogenase, and pyruvate kinase were from Boehringer Mannheim. Enzyme Purification and Preparation of the Phosphorylated Enzyme—Wild type and mutant Dictyostelium NDP kinases were overexpressed in Escherichia coli (XL1-Blue) using plasmid pndk as described previously [33] with small modifications. The enzyme was first preincubated in T buffer (50 mM Tris-HCl, pH 7.5, containing 5 mM MgCl2 and 75 mM KCl) with a saturating amount of [␥-32P]GTP. It was freed of nucleotides by gel filtration on Sephadex G-25. The concentration of the phosphorylated enzyme as well as the absence of nucleotides in the preparation were checked from the absorbance spectrum of the protein. Completeness and R factors refer to the values after using a 2-s cutoff on the reflections
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