Crystal structure and substrate binding mode of ectonucleotide phosphodiesterase/pyrophosphatase-3 (NPP3)

Abstract

Ectonucleotide phosphodiesterase/pyrophosphatase-3 (NPP3) is a membrane-bound glycoprotein that regulates extracellular levels of nucleotides. NPP3 is known to contribute to the immune response on basophils by hydrolyzing ATP and to regulate the glycosyltransferase activity in Neuro2a cells. Here, we report on crystal structures of the nuclease and phosphodiesterase domains of rat NPP3 in complex with different substrates, products and substrate analogs giving insight into details of the catalytic mechanism. Complex structures with a phosphate ion, the product AMP and the substrate analog AMPNPP provide a consistent picture of the coordination of the substrate in which one zinc ion activates the threonine nucleophile whereas the other zinc ion binds the phosphate group. Co-crystal structures with the dinucleotide substrates Ap4A and UDPGlcNAc reveal a binding pocket for the larger leaving groups of these substrates. The crystal structures as well as mutational and kinetic analysis demonstrate that the larger leaving groups interact only weakly with the enzyme such that the substrate affinity is dominated by the interactions of the first nucleoside group. For this moiety, the nucleobase is stacked between Y290 and F207 and polar interactions with the protein are only formed via water molecules thus explaining the limited nucleobase selectivity.

Response regulator PorX coordinates oligonucleotide signalling and gene expression to control the secretion of virulence factors

“…Figure S1, S7) and are probably caused by crystal packing. The pGpG molecule does not fill all pockets of the active site cavity, as also shown in the structures of ENPP1-pApG and ENPP3-Ap4A ( 61 , 62 ) ( Supplementary Figure S6A–C ), suggesting the site accepts larger substrates or allows the reorientation of the ligand during catalysis.…”

“…P. gingivalis produces diguanylate cyclase PGN_1932 ( 58 ) that catalyses the synthesis of c-di-GMP, but it does not possess EAL or HD-GYP homologues, which degrade this cyclic dinucleotide ( 59 , 60 ). Both c-di-GMP and its derivative pGpG would fit in the wide active site of PorX, similar to mouse ENPP1 and rat ENPP3, which were crystallized with pApG and Ap4A, respectively ( 61 , 62 ) ( Supplementary Figure S6 ). Therefore, we attempted to crystallize PorX and inactive variant T272A as complexes with c-di-GMP, pGpG and GMP, to catch any of the three steps of c-di-GMP cleavage, as well as GTP, GTPγS analogue and ATP, by co-crystallization or crystal soaking.…”

“…Table S5). The similarity was high for mouse ENPP1 and rat ENPP3, which have been crystalized with the dinucleotides pApG and Ap4A, respectively ( 61 , 62 ). Like the PglZ domain of PorX, ENPP1 and ENPP3 also feature broad active site clefts with connected pockets.…”

ENPP1’s regulation of extracellular cGAMP is a ubiquitous mechanism of attenuating STING signaling

“…The K237A mutation achieved the opposite of our goal by abolishing ATP degradation but preserving cGAMP degradation. It is possible that K237 is important for stabilizing the β- and γ-phosphates of ATP ( 44 ), but is too far away from the phosphodiester of cGAMP to affect catalysis.…”

Structure and function of the ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family: Tidying up diversity

“…PDE domains have been suggested to support an associative two-step in-line displacement mechanism for catalysis, similar to what was originally described for alkaline phosphatases ( 30 , 31 ). The latter mechanism was supported by crystallographic studies of transition state mimics for ENPP2 ( 29 ) and further backed by additional data for ENPP1 ( 32 ) and ENPP3 ( 33 ). In this model, the catalytic nucleophile is a threonine in all ENPPs (except for ENPP6, where a serine residue is used) and is activated by the Zn2 ion (see Fig.…”