Conserved Polar Residues Stabilize Transmembrane Domains and Promote Oligomerization in Human Nucleoside Triphosphate Diphosphohydrolase 3

145

117

The first comprehensive review of the ubiquitous "ecto-ATPases" by Plesner was published in 1995. A year later, a lymphoid cell activation antigen, CD39, that had been cloned previously, was shown to be an ecto-ATPase. A family of proteins, related to CD39 and a yeast GDPase, all containing the canonical apyrase conserved regions in their polypeptides, soon started to expand. They are now recognized as members of the GDA1_CD39 protein family. Because proteins in this family hydrolyze nucleoside triphosphates and diphosphates, a unifying nomenclature, nucleoside triphosphate diphopshohydrolases (NTPDases), was established in 2000. Membrane-bound NTPDases are either located on the cell surface or membranes of intracellular organelles. Soluble NTPDases exist in the cytosol and may be secreted. In the last 15 years, molecular cloning and functional expression have facilitated biochemical characterization of NTPDases of many organisms, culminating in the recent structural determination of the ecto-domain of a mammalian cell surface NTPDase and a bacterial NTPDase. The first goal of this review is to summarize the biochemical, mutagenesis, and structural studies of the NTPDases. Because of their ability in hydrolyzing extracellular nucleotides, the mammalian cell surface NTPDases (the ecto-NTPDases) which regulate purinergic signaling have received the most attention. Less appreciated are the functions of intracellular NTPDases and NTPDases of other organisms, e.g., bacteria, parasites, Drosophila, plants, etc. The second goal of this review is to summarize recent findings which demonstrate the involvement of the NTPDases in multiple and diverse physiological processes: pathogen-host interaction, plant growth, eukaryote cell protein and lipid glycosylation, eye development, and oncogenesis.

Section: )mentioning

confidence: 86%

“…While evidence for a stable tetrameric NTPDase is generally lacking, Gaddie and Kirley [89] recently provided strong evidence for the existence of a stable dimer for the full-length human NTPDase3. NTPDase3 of many species has a conserved glutamine (Q44 in human NTPDase3) that is located in TMD1 and near the extracellular surface.…”

Section: )mentioning

confidence: 99%

The GDA1_CD39 superfamily: NTPDases with diverse functions

Knowles

2011

145

117

“…Human NTPDase3 was found to preferentially form dimers [138]. Hydrogen bonding involving a conserved glutamine located in TMD1 near the extracellular surface was implicated in dimer formation [141]. The variable oligomeric structure of NTPDases from monomeric to oligomeric forms is in stark contrast to P2X receptors that share similar membrane topology [142].…”

Section: Oligomeric Structurementioning

confidence: 99%

“…Members of the ASKHA structural superfamily are renowned for especially large conformational transitions coupled to function (e.g., Factin versus G-actin). A multitude of experiments have shown that a strong linkage exists between the active site and transmembrane helix interactions [71, 73,125,132,133,141,197]. Oxidative cross-linking experiments carried out with cysteine mutants of membrane-bound variants of NTPDase1 indicate a rather dynamic coupling of coordinated movements of the transmembrane helices to spatial rearrangements of the active site [125,132,133].…”

Section: Active Site and Catalytic Mechanismmentioning

confidence: 99%

“…The transmembrane helices might influence catalysis by changing the frequency or magnitude of the domain motion. Mutational studies suggest that polar residues at the extracellular side of the transmembrane helices and proline-rich linker regions between the ECD and the transmembrane helices participate in the coupling process [132,141,197].…”

Section: Active Site and Catalytic Mechanismmentioning

confidence: 99%

See 1 more Smart Citation

Cellular function and molecular structure of ecto-nucleotidases

Zimmermann

Zebisch

Sträter

2012

888

922

Ecto-nucleotidases play a pivotal role in purinergic signal transmission. They hydrolyze extracellular nucleotides and thus can control their availability at purinergic P2 receptors. They generate extracellular nucleosides for cellular reuptake and salvage via nucleoside transporters of the plasma membrane. The extracellular adenosine formed acts as an agonist of purinergic P1 receptors. They also can produce and hydrolyze extracellular inorganic pyrophosphate that is of major relevance in the control of bone mineralization. This review discusses and compares four major groups of ectonucleotidases: the ecto-nucleoside triphosphate diphosphohydrolases, ecto-5′-nucleotidase, ecto-nucleotide pyrophosphatase/phosphodiesterases, and alkaline phosphatases. Only recently and based on crystal structures, detailed information regarding the spatial structures and catalytic mechanisms has become available for members of these four ecto-nucleotidase families. This permits detailed predictions of their catalytic mechanisms and a comparison between the individual enzyme groups. The review focuses on the principal biochemical, cell biological, catalytic, and structural properties of the enzymes and provides brief reference to tissue distribution, and physiological and pathophysiological functions.

Proline residues link the active site to transmembrane domain movements in human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3)

Gaddie

Kirley

2010

Self Cite

The active sites of the membrane-bound nucleoside triphosphate diphosphohydrolases (NTPDases) regulate and are regulated by coordinated and spatially distant movements of their transmembrane helices, modulating enzyme activity, and substrate specificity. Using sitedirected mutagenesis, the roles of the conserved proline residues (N-terminal: P52 and P53; C-terminal: P472, P476, P481, P484, and P485) of human NTPDase3, located in the "linker regions" that connect the N-and C-terminal transmembrane helices with the extracellular active site, were examined. Single cysteine substitutions were strategically placed in the transmembrane domain (N-terminal helix: V42C; C-terminal helix: G489C) to serve as crosslinking "sensors" of helical interactions. These "sensor" background mutant proteins (V42C and G489C NTPDase3) are enzymatically active and are cross-linked by copper phenanthroline less efficiently in the presence of adenosine triphosphate (ATP). Proline to alanine substitutions at P53, P481, P484, and P485 in the V42C background, as well as P53, P481, and P484 in the G489C background, exhibited decreased nucleotidase activities. More importantly, alanine substitutions at P53 and P481 in the V42C background and P481 in the G489C background no longer exhibited the ATP-induced decrease in transmembrane cross-linking efficiency. Interestingly, the P485A mutation abolished oxidative cross-linking at G489C both in the presence and absence of ATP. Taken together, these results suggest a role for proline residues 53 and 481 in the linker regions of human NTPDase3 for coupling nucleotide binding at the enzyme active site to movements and/or rearrangements of the transmembrane helices necessary for optimal nucleotide hydrolysis.