Structure and function of ecto-nucleotidases in purinergic signaling

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In addition to its important cellular function in metabolism, ATP also serves as an extracellular signaling substance. Extracellular nucleotides influence a wide variety of short-term (acute) physiological processes, including exocrine and endocrine secretion, immune responses, inflammation, nociceptive mechanosensory transduction, platelet aggregation and endothelial-mediated vasodilatation. Long-term (trophic) processes affected are cell proliferation, differentiation, migration and death as for example in development, regeneration and cancer.Extracellular nucleotidases are involved in the metabolism of the nucleotides. The NTPDases (ecto-nucleoside triphosphate diphosphohydrolases) dephosphorylate ATP via ADP to AMP. NTPDase1, -2, -3 and -8 face the extracellular space and function in purinergic signal transmission whereas the NTPDases 4-7 are located in intracellular organelles such as the Golgi. Here they are involved in protein glycosylation and convert NDPs to NMPs to avoid inhibition of glycosyl transferases and to provide NMPs for exchange against NDP-sugars by an antiporter. In addition to the NTPDases, nucleotide pyrophosphatases/phosphodiesterases (NPPs) are involved in extracellular nucleotide metabolism.The aim of the project is to characterize the molecular structures and catalytic mechanisms of NTPDases and NPPs by X-ray crystallography, mutagenesis studies and activity assays. A major goal is the first structure determination of an intracellular NTPDase involved in protein glycosylation. The active site structures and unique substrate specificity of these enzymes cannot be understood based on homology models generated from the available structures of NTPDase1 and 2. To this end, we will determine crystal structures of human and rat NTPDases 4-7. Co-crystal structures with substrates and inhibitors will define the substrate binding interactions and the molecular basis for the differences in substrate specificity (NDP vs. NTP and for different nucleobases). NTPDase1 hydrolyses ATP to AMP without the release of ADP. It was previously shown that this processivity depends on the membrane anchoring of the enzyme. Via mutagenesis studies we will test the hypothesis that a long hydrophobic conserved loop in the extracellular NTPDases is involved in membrane anchoring and mediates the coupling between the transmembrane part and the ectodomain. In addition, we will determine crystal structures of the yet uncharacterized ecto-NTPDases 3 and 8. Another project part concerns the specificity and mechanism of NPPs. We were able to crystallize rat NPP3. We will determine the X-ray structure of the enzyme and analyze co-crystal structures with various nucleotide substrates and products to understand the differences in substrate specificity between various NPPs involved in purinergic signaling and calcification. In collaboration we will aid the development of specific inhibitors of these enzymes as biological and pharmaceutical tools.