Structures of Legionella pneumophila NTPDase1 in complex with polyoxometallates.

Nucleoside triphosphate diphosphohydrolases (NTPDases) are secreted or membrane-bound ectonucleotidases that hydrolyze the anhydride bonds of nucleoside triphosphates and nucleoside diphosphates. Mammalian cell-surface NTPDase enzymes are inhibited by various polyoxometallates. Here, the structures of NTPDase1 from the bacterium Legionella pneumophila (LpNTPDase1) in complex with the dodecatungstate POM-1, decavanadate and octamolybdate/heptamolybdate are described. The metal clusters are bound at different sites but always in a highly ordered fashion via electrostatic interactions and hydrogen bonds. For octamolybdate, covalent interactions after oxygen ligand exchange by a serine and histidine side chain are also observed. The potential inhibitory mechanism and the use of the metal clusters as phasing tools for new NTPDase structures are discussed. The binding mode of a tartrate ion at the catalytic centre suggests novel strategies for the structure-based design of NTPDase inhibitors, and the observation of the enzyme in an intermediate open state contributes to our understanding of NTPDase enzyme dynamics.

Structural insight into activation mechanism of Toxoplasma gondii nucleoside triphosphate diphosphohydrolases by disulfide reduction.

The intracellular parasite Toxoplasma gondii produces two nucleoside triphosphate diphosphohydrolases (NTPDase1 and -3). These tetrameric, cysteine-rich enzymes require activation by reductive cleavage of a hitherto unknown disulfide bond. Despite a 97% sequence identity, both isozymes differ largely in their ability to hydrolyze ATP and ADP. Here, we present crystal structures of inactive NTPDase3 as an apo form and in complex with the product AMP to resolutions of 2.0 and 2.2 Å, respectively. We find that the enzyme is present in an open conformation that precludes productive substrate binding and catalysis. The cysteine bridge 258-268 is identified to be responsible for locking of activity. Crystal structures of constitutively active variants of NTPDase1 and -3 generated by mutation of Cys(258)-Cys(268) show that opening of the regulatory cysteine bridge induces a pronounced contraction of the whole tetramer. This is accompanied by a 12° domain closure motion resulting in the correct arrangement of all active site residues. A complex structure of activated NTPDase3 with a non-hydrolyzable ATP analog and the cofactor Mg(2+) to a resolution of 2.85 Å indicates that catalytic differences between the NTPDases are primarily dictated by differences in positioning of the adenine base caused by substitution of Arg(492) and Glu(493) in NTPDase1 by glycines in NTPDase3.

Crystallographic snapshots along the reaction pathway of nucleoside triphosphate diphosphohydrolases.

In vertebrates, membrane-bound ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) on the cell surface are responsible for signal conversion and termination in purinergic signaling by extracellular nucleotides. Here we present apo and complex structures of the rat NTPDase2 extracellular domain and Legionella pneumophila NTPDase1, including a high-resolution structure with a transition-state analog. Comparison of ATP and ADP binding modes shows how NTPDases engage the same catalytic site for hydrolysis of nucleoside triphosphates and diphosphates. We find that this dual specificity is achieved at the expense of base specificity. Structural and mutational studies indicate that a conserved active-site water is replaced by the phosphate product immediately after phosphoryl transfer. Partial base specificity for purines in LpNTPDase1 is based on a different intersubunit base binding site for pyrimidine bases. A comparison of the bacterial enzyme in six independent crystal forms shows that NTPDases can undergo a domain closure motion of at least 17°.

Crystallographic evidence for a domain motion in rat nucleoside triphosphate diphosphohydrolase (NTPDase) 1.

Nucleoside triphosphate diphosphohydrolases (NTPDases) are a physiologically important class of membrane-bound ectonucleotidases responsible for the regulation of extracellular levels of nucleotides. CD39 or NTPDase1 is the dominant NTPDase of the vasculature. By hydrolyzing proinflammatory ATP and platelet-activating ADP to AMP, it blocks platelet aggregation and supports blood flow. Thus, great interest exists in understanding the structure and dynamics of this prototype member of the eukaryotic NTPDase family. Here, we report the crystal structure of a variant of soluble NTPDase1 lacking a putative membrane interaction loop identified between the two lobes of the catalytic domain. ATPase and ADPase activities of this variant are determined via a newly established kinetic isothermal titration calorimetry assay and compared to that of the soluble NTPDase1 variant characterized previously. Complex structures with decavanadate and heptamolybdate show that both polyoxometallates bind electrostatically to a loop that is involved in binding of the nucleobase. In addition, a comparison of the domain orientations of the four independent proteins in the crystal asymmetric unit provides the first direct experimental evidence for a domain motion of NTPDases. An interdomain rotation angle of up to 7.4° affects the active site cleft between the two lobes of the protein. Comparison with a previously solved bacterial NTPDase structure indicates that the domains may undergo relative rotational movements of more than 20°. Our data support the idea that the influence of transmembrane helix dynamics on activity is achieved by coupling to a domain motion.