Ectonucleotide phosphodiesterase/pyrophosphatase-3 (PDB ID: 6F33)(NPP3) from Rattus norvegicus is a monomeric membrane-bound nucleotide hydrolase that hydrolyzes extracellular pyrophosphate or phosphodiester bonds and acts as a regulator in glycosyltransferase activity by regulating extracellular levels of nucleotides (1,3). A pyrophosphatase is an enzyme that catalyzes the hydrolysis of inorganic pyrophosphate to two molecules of inorganic phosphate. NPP3 is known to negatively regulates chronic allergic inflammation and is rapidly expressed on mast cells and basophils. Its regulation of ATP suppresses mast cell and basophil activity helping control allergic and chronic inflammation. These properties make NPP3 a viable activation marker for allergic diseases (2). NPP3 is highly expressed in many cancer cells including acute basophilic leukemia, renal cell carcinoma, and clear cell renal cell carcinoma and has been identified as a potential human cancer specific-antigen for cancer therapy through suppression subtractive hybridization (3). It also plays a key role in the regulation of N-Acetylglucosaminyltransferase IX (GnT-IX), a brain-specific glycosyltransferase that synthesizes branched O-mannose, in Neuro2a cells mediated by the hydrolysis of nucleotide sugar donor substrates linking it directly to glycosyltransferase activity and the modulation of cellular glycosylation profiles (4,13). 

      NPP3 belongs to the NPP glycoprotein family, which consists of seven structurally similar proteins. Named NPP1-NPP7, these proteins share the ability to hydrolyze pyrophosphate or phosphodiester bonds. Similar to NPP3, NPP1 and NPP4 hydrolyze nucleotides. NPP2 (autotaxin) hydrolyzes lysophosphatidylcholine (LPC) producing lysophosphatidic acid (LPA) allowing it to activate G-protein-coupled receptors. Although the specific role of NPP5 is unknown, it is able to cleave adenine dinucleotides suggesting a role in NAD-based neurotransmission. NPP6 hydrolyzes glycerophosphocholine (GPC), and NPP7 controls cholesterol levels by converting sphingomyelin. All NPP proteins share a zinc-binding catalytic domain (1,14). 

NPP3 contains two somatomedin B domains, one type I phosphodiesterase/nucleotide pyrophosphatase domain (residues 140-544) and one DNA/RNA non-specific endonuclease (residues 563-875) (1,9). It has a molecular weight of 85.3 KDa, and an isoelectric point of 6.16 (8). The reported crystalized structure of NPP3 is a homodimer (A and B chains) in complex with the substrate analog adenosine-5?-[(alpha,beta)-imido]triphosphate (AMPNPP). Dimerization of NPP3 in cells is most likely mediated by transmembrane helix interaction similar to that of the dimerization of NPP1 (1). In order to achieve the crystal structure, an inactive mutant of NPP3 was created by changing the Thr-206 nucleophile to Ala-206 allowing it to bind to AMPNPP as the non-mutant NPP3 resulted in the observation of the hydrolyzed product AMP in its active site (1). 

The crystals of the NPP3 homodimer formed an interaction interface of 536 Å2 and diffracted to 2.4Å (1). The crystal structure was obtained by X-Ray diffraction of crystals formed using the vapor diffusion hanging drop method. The crystallization was carried out at a pH of 6, a temperature of 292.0K, and with the following conditions: 0.2M KCl, 0.1M MgAcetate, 0.05 NaCacodylate, 10.2% PEG8000, 1mM CaCl2, and 0.1 mM ZnSO4 (9). The ligand 5'-O-[(S)-hydroxy{[(S)-hydroxy(phosphonooxy)phosphoryl]amino}phosphoryl]adenosine (AMP-PNP) is present in the crystalized structure. AMP-PNP is a non-hydrolyzable ATP analog that inhibits ATP-dependent enzyme systems. When bound to a protein, this ligand competes for and prohibits any process involving hydrolysis of ATP (9). The crystalized structure of NPP3 is missing 139 residues from its N terminus, 4 residues from its C terminus, 2 residues from its phosphodiesterase domain, and 9 other residues throughout the remainder of the structure. The 139 missing residues from the N terminus include both somatomedin B domains (9). 

NPP3 is comprised of 53% random coil, 26% alpha helix, 17% beta sheet, and 4% beta turn (9). The phosphodiesterase/nucleotide pyrophosphatase domain and the endonuclease domain interact over an interface of 1279 Å2. These two domains are linked and stabilized via five salt bridges, 16 hydrogen bond interactions, a loop of 18 residues, and a disulfide bridge from Cys-429 to Cys-818. NPP3 contains a "WPG loop" of residues 272-290 within its catalytic domain preventing the formation of a hydrophobic pocket for binding lysophospholipids allowing substrate specificity in the active site (1). A glycan chain at Asn-533 forms a hydrogen bond with His-758 coordinating a Ca2+ ion of the EF-hand like motif (1). An EF-hand motif is a helix-loop-helix structure found in calcium-binding proteins (9). EF-hand motifs are required for many cellular processes, associated with cancer, autism, cardiac arrhythmias, skeletal muscle diseases, neuronal diseases, and Alzheimer?s. These motifs are control centers for the regulation of calcium ions that influence the signaling and regulatory roles within cells (6). The EF-hand like motif in NPP3 is composed of residues Asp-572, Asn-754,Asp-756 and Asp-760. A similar calcium binding structure is conserved in NPP1 and NPP2. In addition to its biological importance, this EF-hand like motif is important for the stabilization of the nuclease domain and for the protein?s catalytic activity (1). Asn-533 is NPP3's major glycosylation site. Although the type of glycosylation reactions are unknown, an additional 6 glycosylation sites were identified by electron density maps at residues Asn-237, Asn-280, Asn-289, Asn-574, Asn-702, and Asn-789. Only Asn-533, Asn-237, Asn-280, and Asn-289 were glycosylated with N-acetyl-Glucosamine in the crystal structure. An glycosylation site was predicted at Asn-594 but was not observed for the A chain possibly due to the flexibility of the loop region. Monomer B displays clear glycosylation affinity at Asn-594 but is not glycosylated in the crystal structure (1). 

NPP3 also contains N-Acetyl-D-glucosamine in conjugation with the Thr-482 and His-330 side chains while Ser-237 forms a hydrogen bond with the sugar's hydroxyl group and Lys-205 forms van-der-walls contacts with the sugar (not shown in the crystal structure)(1). 

The active site of NPP3 contains two Zn2+ ions which are coordinated by Asp-168, Thr-206, Asp-326, His-330, Asp-373, His-374, and His-483. These residues are conserved in the NPP Family within the phosphodiesterase/pyrophosphatase domain. A phosphate ion is coordinated to the first zinc atom via an oxygen atom in addition to its hydrogen bonding to Thr-206 (Shown as Ala-206 in the crystal structure). This zinc atom has a distorted five-fold coordination whereas the second zinc atom is coordinated tetrahedrally (1). The side chain of Asn-227 is within hydrogen bond distance to a phosphate oxygen but the hydrogen bond angle of 113.4° suggests a very weak interaction (1). AMP-PNP is contained within a GGXXG motif ligand binding pocket from residues Gly-480 to Gly-484 and is involved in pi-pi stacking interactions between Phe-207 and Tyr-290 (1). 

Using PSI-BLAST, an online program that compares primary structure of a query protein to known proteins, where an E value of less than 0.05 is considered to have high primary structure similarity to the query protein, Ectonucleotide pyrophosphatase/phosphodiesterase-1 (PDB ID: 4B56)(NPP1) from Mus musculus returned an E value of 0.00 when compared to NPP3. PSI-BLAST indicated 49% identity between the A chain of NPP3 and the A chain of NPP1 with 67% positive alignment and 1% structure gap (12). Using the Dali server, a utility that compares conserved tertiary structures between two proteins where a score greater than 2 indicates significantly similar structure, NPP1 and NPP3 presented a Z score of 62.9 (7). These bioinformatic tools show NPP1 and NPP3 have similar structures, and as they both belong to the NPP family, they also have similar functions. 

NPP1 is a nucleotide hydrolase that acts through the conserved active site zinc ion coordinating residues also present in NPP3 and is a key regulator of tissue calcification and one development through its generation of inorganic phosphate. A lack of NPP1 is an underling cause to calcification disorders such as abnormal bone development and the hardening of soft tissues such as ligaments, tendons, and arterial smooth muscle walls. These problems are life-threatening in infancy and early childhood. Overexpression of NPP1 can also lead to apoptosis-mediated calcification of the aortic valves (5). 

Similar to NPP3, NPP1 exists within cells as a monomer but has been shown to form homodimers through intermembrane disulfide bonding. NPP1 contains three conserved cysteines in its transmembrane domain. The combined mutagenesis of these residues prevents dimer formation (5). The dimer interface area of NPP1 is around 1,540 Å2 and is linked by 14 hydrogen bonds and 5 salt bridges (NPP3: 1,279 Å2, 16 Hydrogen bonds, and 5 salt bridges) making it larger than NPP3 by 261 Å2 with two fewer hydrogen bonds (5). 

In contrast to NPP3, the somatomedin B domains are conserved in the crystal structure of NPP1 and the destruction of the second monomer?s somatomedin B domains may be caused by solvent disordering during the crystallization experiment (5). NPP1 was crystalized with two monosaccharides, alpha-D-mannose and beta-D-mannose that the crystal structure of NPP3 does not possess. NPP3 was crystalized with AMP-PNP which was not in the crystal structure of NPP1. They both contain Zn2+ and Ca2+ ions, and the C-2 N-acetylated monosaccharide N-acetyl-D-glucosamine (9). The reported crystalized structure of NPP1 is comprised of 48% random coil, 28% alpha helix, 17% beta sheet, and 7% beta turn which is very similar to the secondary of NPP3. The crystal structure of the A chain of NPP1 conserved its somatomedin B domains consisting of 80 amino acids, most of which is mostly random coil containing four small loops of alpha helices, and one tight turn (9). 

While the crystal structure of NPP3 elucidates many of its structural features, a fair amount of how its structure brings rise to its function has yet to be uncovered. NPP3 plays a role in the cellular glycosylation profile and has the potential to be an even greater factor for clinical relevance including chronic allergy therapy and cancer treatments.