Specific Mutations Reverse Regulatory Effects of Adenosine Phosphates and Increase Their Binding Stoichiometry in CBS Domain-Containing Pyrophosphatase.

Regulatory cystathionine ß-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of the underlying regulation mechanism. Tetrameric microbial inorganic pyrophosphatase containing such domains (CBS-PPase) is allosterically inhibited by AMP and ADP and activated by ATP and cell alarmones diadenosine polyphosphates. Each CBS-PPase subunit contains a pair of CBS domains but binds cooperatively to only one molecule of the mono-adenosine derivatives. We used site-directed mutagenesis of Desulfitobacterium hafniense CBS-PPase to identify the key elements determining the direction of the effect (activation or inhibition) and the "half-of-the-sites" ligand binding stoichiometry. Seven amino acid residues were selected in the CBS1 domain, based on the available X-ray structure of the regulatory domains, and substituted by alanine and other residues. The interaction of 11 CBS-PPase variants with the regulating ligands was characterized by activity measurements and isothermal titration calorimetry. Lys100 replacement reversed the effect of ADP from inhibition to activation, whereas Lys95 and Gly118 replacements made ADP an activator at low concentrations but an inhibitor at high concentrations. Replacement of these residues for alanine increased the stoichiometry of mono-adenosine phosphate binding by twofold. These findings identified several key protein residues and suggested a "two non-interacting pairs of interacting regulatory sites" concept in CBS-PPase regulation.

Coupling enzymatic activity and gating in an ancient TRPM chanzyme and its molecular evolution.

Channel enzymes represent a class of ion channels with enzymatic activity directly or indirectly linked to their channel function. We investigated a TRPM2 chanzyme from choanoflagellates that integrates two seemingly incompatible functions into a single peptide: a channel module activated by ADP-ribose with high open probability and an enzyme module (NUDT9-H domain) consuming ADP-ribose at a remarkably slow rate. Using time-resolved cryogenic-electron microscopy, we captured a complete series of structural snapshots of gating and catalytic cycles, revealing the coupling mechanism between channel gating and enzymatic activity. The slow kinetics of the NUDT9-H enzyme module confers a self-regulatory mechanism: ADPR binding triggers NUDT9-H tetramerization, promoting channel opening, while subsequent hydrolysis reduces local ADPR, inducing channel closure. We further demonstrated how the NUDT9-H domain has evolved from a structurally semi-independent ADP-ribose hydrolase module in early species to a fully integrated component of a gating ring essential for channel activation in advanced species.

Structural and biochemical characterization of active sites mutant in human inorganic pyrophosphatase.

Inorganic pyrophosphatases (PPases) are enzymes that catalyze the conversion of inorganic pyrophosphate (PPi) into phosphate (Pi). Human inorganic pyrophosphatase 1 (Hu-PPase) exhibits high expression levels in a variety of tumors and plays roles in cell proliferation, apoptosis, invasion and metastasis, making it a promising prognostic biomarker and a target for cancer therapy. Despite its widespread presence, the catalytic mechanism of Hu-PPase in humans remains inadequately understood. The signature motif amino acid sequence (DXDPXD) within the active sites of PPases is preserved across different species. In this research, an enzymatic activity assay revealed that mutations led to a notable reduction in enzymatic function, although the impact of the four amino acids on the activity of the pocket varied. To investigate the influence of these residues on the substrate binding and enzymatic function of PPase, the crystal structure of the Hu-PPase-ED quadruple mutant (D116A/D118A/P119A/D121A) was determined at 1.69 Å resolution. The resulting structure maintained a barrel-like shape similar to that of the wild-type, albeit lacking Mg2+ ions. Molecular docking analysis demonstrated a decreased ability of Hu-PPase-ED to bind to PPi. Further, molecular dynamics simulation analysis indicated that the mutation rendered the loop of Mg2+ ion-binding residues less stable. Therefore, the effect on enzyme activity did not result from a change in the gross protein structure but rather from a mutation that abolished the Mg2+-coordinating groups, thereby eliminating Mg2+ binding and leading to the loss of enzyme activity.

Differential effects of the lipidic and ionic microenvironment on NPP1's phosphohydrolase and phosphodiesterase activities.

Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) is an enzyme present in matrix vesicles (MV). NPP1 participates on the regulation of bone formation by producing pyrophosphate (PPi) from adenosine triphosphate (ATP). Here, we have used liposomes bearing dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (SM), and cholesterol (Chol) harboring NPP1 to mimic the composition of MV lipid rafts to investigate ionic and lipidic influence on NPP1 activity and mineral propagation. Atomic force microscopy (AFM) revealed that DPPC-liposomes had spherical and smooth surface. The presence of SM and Chol elicited rough and smooth surface, respectively. NPP1 insertion produced protrusions in all the liposome surface. Maximum phosphodiesterase activity emerged at 0.082 M ionic strength, whereas maximum phosphomonohydrolase activity arose at low ionic strength. Phosphoserine-Calcium Phosphate Complex (PS-CPLX) and amorphous calcium-phosphate (ACP) induced mineral propagation in DPPC- and DPPC:SM-liposomes and in DPPC:Chol-liposomes, respectively. Mineral characterization revealed the presence of bands assigned to HAp in the mineral propagated by NPP1 harbored in DPPC-liposomes without nucleators or in DPPC:Chol-liposomes with ACP nucleators. These data show that studying how the ionic and lipidic environment affects NPP1 properties is important, especially for HAp obtained under controlled conditions in vitro.

Mammalian Nudt15 hydrolytic and binding activity on methylated guanosine mononucleotides.

The Nudt15 enzyme of the NUDIX protein family is the subject of extensive study due to its action on thiopurine drugs used in the treatment of cancer and inflammatory diseases. In addition to thiopurines, Nudt15 is enzymatically active in vitro on several nucleotide substrates. It has also been suggested that this enzyme may play a role in 5'RNA turnover by hydrolyzing m7GDP, a product of mRNA decapping. However, no detailed studies on this substrate with Nudt15 are available. Here, we analyzed the enzymatic activity of Nudt15 with m7GDP, its triphosphate form m7GTP, and the trimethylated counterparts (m32,2,7GDP and m32,2,7GTP). Kinetic data revealed a moderate activity of Nudt15 toward these methylated mononucleotides compared to the dGTP substrate. However m7GDP and m32,2,7GDP showed a distinct stabilization of Nudt15 upon ligand binding, in the same range as dGTP, and thus these two mononucleotides may be used as leading structures in the design of small molecule binders of Nudt15.

Protonation states of Asp residues in the human Nudix hydrolase MTH1 contribute to its broad substrate recognition.

Human MutT homolog 1 (MTH1), also known as Nudix-type motif 1 (NUDT1), hydrolyzes 8-oxo-dGTP and 2-oxo-dATP with broad substrate recognition and has attracted attention in anticancer therapeutics. Previous studies on MTH1 have proposed that the exchange of the protonation state between Asp119 and Asp120 is essential for the broad substrate recognition of MTH1. To understand the relationship between protonation states and substrate binding, we determined the crystal structures of MTH1 at pH 7.7-9.7. With increasing pH, MTH1 gradually loses its substrate-binding ability, indicating that Asp119 is deprotonated at pH 8.0-9.1 in 8-oxo-dGTP recognition and Asp120 is deprotonated at pH 8.6-9.7 in 2-oxo-dATP recognition. These results confirm that MTH1 recognizes 8-oxo-dGTP and 2-oxo-dATP by exchanging the protonation state between Asp119 and Asp120 with higher pKa .

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

Ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family members (ENPP1-7) have been implicated in key biological and pathophysiological processes, including nucleotide and phospholipid signaling, bone mineralization, fibrotic diseases, and tumor-associated immune cell infiltration. ENPPs are single-pass transmembrane ecto-enzymes, with notable exceptions of ENPP2 (Autotaxin) and ENNP6, which are secreted and glycosylphosphatidylinositol (GPI)-anchored, respectively. ENNP1 and ENNP2 are the best characterized and functionally the most interesting members. Here, we review the structural features of ENPP1-7 to understand how they evolved to accommodate specific substrates and mediate different biological activities. ENPPs are defined by a conserved phosphodiesterase (PDE) domain. In ENPP1-3, the PDE domain is flanked by two N-terminal somatomedin B-like domains and a C-terminal inactive nuclease domain that confers structural stability, whereas ENPP4-7 only possess the PDE domain. Structural differences in the substrate-binding site endow each protein with unique characteristics. Thus, ENPP1, ENPP3, ENPP4, and ENPP5 hydrolyze nucleotides, whereas ENPP2, ENPP6, and ENNP7 evolved as phospholipases through adaptions in the catalytic domain. These adaptations explain the different biological and pathophysiological functions of individual members. Understanding the ENPP members as a whole advances our insights into common mechanisms, highlights their functional diversity, and helps to explore new biological roles.

Analysis of ligand binding and resulting conformational changes in pyrophosphatase NUDT9.

Nudix hydrolase 9 (NUDT9) is a member of the nucleoside linked to another moiety X (NUDIX) protein superfamily, which hydrolyses a broad spectrum of organic pyrophosphates from metabolic processes. ADP-ribose (ADPR) has been the only known endogenous substrate accepted by NUDT9 so far. The Ca2+ -permeable transient receptor potential melastatin subfamily 2 (TRPM2) channel contains a homologous NUDT9-homology (NUDT9H) domain and is activated by ADPR. Sustained Ca2+ influx via ADPR-activated TRPM2 triggers apoptotic mechanisms. Thus, a precise regulation of cellular ADPR levels by NUDT9 is essential. A detailed characterization of the enzyme-substrate interaction would help to understand the high substrate specificity of NUDT9. Here, we analysed ligand binding to NUDT9 using a variety of biophysical techniques. We identified 2'-deoxy-ADPR as an additional substrate for NUDT9. Similar enzyme kinetics and binding affinities were determined for the two ligands. The high-affinity binding was preserved in NUDT9 containing the mutated NUDIX box derived from the human NUDT9H domain. NMR spectroscopy indicated that ADPR and 2'-deoxy-ADPR bind to the same binding site of NUDT9. Backbone resonance assignment and subsequent molecular docking allowed further characterization of the binding pocket. Substantial conformational changes of NUDT9 upon ligand binding were observed which might allow for the development of NUDT9-based ADPR fluorescence resonance energy transfer sensors that may help with the analysis of ADPR signalling processes in cells in the future.

Structural and mechanistic insights into the bifunctional HISN2 enzyme catalyzing the second and third steps of histidine biosynthesis in plants.

The second and third steps of the histidine biosynthetic pathway (HBP) in plants are catalyzed by a bifunctional enzyme-HISN2. The enzyme consists of two distinct domains, active respectively as a phosphoribosyl-AMP cyclohydrolase (PRA-CH) and phosphoribosyl-ATP pyrophosphatase (PRA-PH). The domains are analogous to single-domain enzymes encoded by bacterial hisI and hisE genes, respectively. The calculated sequence similarity networks between HISN2 analogs from prokaryotes and eukaryotes suggest that the plant enzymes are closest relatives of those in the class of Deltaproteobacteria. In this work, we obtained crystal structures of HISN2 enzyme from Medicago truncatula (MtHISN2) and described its architecture and interactions with AMP. The AMP molecule bound to the PRA-PH domain shows positioning of the N1-phosphoribosyl relevant to catalysis. AMP bound to the PRA-CH domain mimics a part of the substrate, giving insights into the reaction mechanism. The latter interaction also arises as a possible second-tier regulatory mechanism of the HBP flux, as indicated by inhibition assays and isothermal titration calorimetry.

Crystal structures of NUDT15 variants enabled by a potent inhibitor reveal the structural basis for thiopurine sensitivity.

The enzyme NUDT15 efficiently hydrolyzes the active metabolites of thiopurine drugs, which are routinely used for treating cancer and inflammatory diseases. Loss-of-function variants in NUDT15 are strongly associated with thiopurine intolerance, such as leukopenia, and preemptive NUDT15 genotyping has been clinically implemented to personalize thiopurine dosing. However, understanding the molecular consequences of these variants has been difficult, as no structural information was available for NUDT15 proteins encoded by clinically actionable pharmacogenetic variants because of their inherent instability. Recently, the small molecule NUDT15 inhibitor TH1760 has been shown to sensitize cells to thiopurines, through enhanced accumulation of 6-thio-guanine in DNA. Building upon this, we herein report the development of the potent and specific NUDT15 inhibitor, TH7755. TH7755 demonstrates a greatly improved cellular target engagement and 6-thioguanine potentiation compared with TH1760, while showing no cytotoxicity on its own. This potent inhibitor also stabilized NUDT15, enabling analysis by X-ray crystallography. We have determined high-resolution structures of the clinically relevant NUDT15 variants Arg139Cys, Arg139His, Val18Ile, and V18_V19insGlyVal. These structures provide clear insights into the structural basis for the thiopurine intolerance phenotype observed in patients carrying these pharmacogenetic variants. These findings will aid in predicting the effects of new NUDT15 sequence variations yet to be discovered in the clinic.

The Asp1 pyrophosphatase from S. pombe hosts a [2Fe-2S]2+ cluster in vivo.

The Schizosaccharomyces pombe Asp1 protein is a bifunctional kinase/pyrophosphatase that belongs to the highly conserved eukaryotic diphosphoinositol pentakisphosphate kinase PPIP5K/Vip1 family. The N-terminal Asp1 kinase domain generates specific high-energy inositol pyrophosphate (IPP) molecules, which are hydrolyzed by the C-terminal Asp1 pyrophosphatase domain (Asp1365-920). Thus, Asp1 activities regulate the intracellular level of a specific class of IPP molecules, which control a wide number of biological processes ranging from cell morphogenesis to chromosome transmission. Recently, it was shown that chemical reconstitution of Asp1371-920 leads to the formation of a [2Fe-2S] cluster; however, the biological relevance of the cofactor remained under debate. In this study, we provide evidence for the presence of the Fe-S cluster in Asp1365-920 inside the cell. However, we show that the Fe-S cluster does not influence Asp1 pyrophosphatase activity in vitro or in vivo. Characterization of the as-isolated protein by electronic absorption spectroscopy, mass spectrometry, and X-ray absorption spectroscopy is consistent with the presence of a [2Fe-2S]2+ cluster in the enzyme. Furthermore, we have identified the cysteine ligands of the cluster. Overall, our work reveals that Asp1 contains an Fe-S cluster in vivo that is not involved in its pyrophosphatase activity.

Development of a High-Throughput Assay to Identify Inhibitors of ENPP1.

The innate immune response to cancer is initiated by cytosolic DNA, where it binds to cGAS and triggers type I interferon (IFN) expression via the STING receptor, leading to activation of tumor-specific T cells. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as the primary enzyme responsible for degrading cGAMP, and therefore it is under intense investigation as a therapeutic target for cancer immunotherapy. ENPP1 hydrolyzes cGAMP to produce AMP and GMP, and hydrolyzes ATP and other nucleotides to monophosphates and pyrophosphate. We developed a robust, high-throughput screening (HTS)-compatible enzymatic assay method for ENPP1 using the Transcreener AMP2/GMP2 Assay, a competitive fluorescence polarization (FP) immunoassay that enables direct detection of AMP and GMP in a homogenous format. The monoclonal antibody used in the Transcreener AMP2/GMP2 Assay showed more than 104-fold selectivity for AMP and GMP versus cGAMP, and 3000-fold selectivity for AMP over ATP, indicating that the assay can be used for detection at initial velocity with either substrate. A working concentration of 100 pM ENPP1 was determined as optimal with a 60 min reaction period, enabling screening with very low quantities of enzyme. A Z' value of 0.72 was determined using ATP as substrate, indicating a high-quality assay. Consistent with previous studies, we found that ENPP1 preferred ATP as a substrate when compared with other nucleotides like GTP, ADP, and GDP. ENPP1 showed a 20-fold selectivity for 2'3'cGAMP compared with 2'3'c-diGMP and showed no activity with 3'3'c-diAMP. The Transcreener AMP2/GMP2 Assay should prove to be a valuable tool for the discovery of ENPP1 lead molecules.

ADP-ribose and analogues bound to the deMARylating macrodomain from the bat coronavirus HKU4.

Macrodomains are proteins that recognize and hydrolyze ADP ribose (ADPR) modifications of intracellular proteins. Macrodomains are implicated in viral genome replication and interference with host cell immune responses. They are important to the infectious cycle of Coronaviridae and Togaviridae viruses. We describe crystal structures of the conserved macrodomain from the bat coronavirus (CoV) HKU4 in complex with ligands. The structures reveal a binding cavity that accommodates ADPR and analogs via local structural changes within the pocket. Using a radioactive assay, we present evidence of mono-ADPR (MAR) hydrolase activity. In silico analysis presents further evidence on recognition of the ADPR modification for hydrolysis. Mutational analysis of residues within the binding pocket resulted in diminished enzymatic activity and binding affinity. We conclude that the common structural features observed in the macrodomain in a bat CoV contribute to a conserved function that can be extended to other known macrodomains.

Purification and preliminary characterization of four Rel homologues from pathogenic bacteria: Implications for species-specific inhibitor design.

Resistance to antibiotics is a serious concern to treat infectious diseases and also, for food preservation. Existing antibiotics generally inhibit enzymes participating in key bacterial processes, such as formation of cell wall, replication, transcription and translation. However, bacteria have rapidly evolved new mechanisms to combat these antibiotics and it hence becomes indispensable to identify newer targets and identify/design inhibitors against them. Another concern is that most antibiotics are broad spectrum; they largely bind and inhibit the active site of the target enzyme. Rel proteins, which synthesize (and hydrolyze) (p)ppGpp in response to a variety of stress encountered by bacteria, is a profitable target owing to its distinct absence in humans and an intricate regulation of the catalytic activities. Inactivation of (p)ppGpp synthesis by Rel, disables bacterial survival in Mycobacterium tuberculosis and Staphylococcus aureus, while inactivating the hydrolysis activity was lethal. The poor MIC values of the currently known Rel inhibitors present a distinct opportunity to develop better inhibitors and warrants a detailed structural characterization and understanding of the complex regulation in Rel proteins. It will open new avenues for the design of effective, species-specific inhibitors. In an attempt to identify unique sites for inhibitor design using structure-based approaches, we initiate a study of Rel homologues from four different pathogenic bacteria, in order to compare their attributes with well characterized Rel homologues. Here, we present cloning, over-expression, purification and preliminary characterization of these four homologues; and suggest similarities and differences that can be exploited for inhibitor design.

Structural comparisons of host and African swine fever virus dUTPases reveal new clues for inhibitor development.

African swine fever, caused by the African swine fever virus (ASFV), is among the most significant swine diseases. There are currently no effective treatments against ASFV. ASFV contains a gene encoding a dUTPase (E165R), which is required for viral replication in swine macrophages, making it an attractive target for inhibitor development. However, the full structural details of the ASFV dUTPase and those of the comparable swine enzyme are not available, limiting further insights. Herein, we determine the crystal structures of ASFV dUTPase and swine dUTPase in both their ligand-free and ligand-bound forms. We observe that the swine enzyme employs a classical dUTPase architecture made up of three-subunit active sites, whereas the ASFV enzyme employs a novel two-subunit active site. We then performed a comparative analysis of all dUTPase structures uploaded in the Protein Data Bank (PDB), which showed classical and non-classical types were mainly determined by the C-terminal ß-strand orientation, and the difference was mainly related to the four amino acids behind motif IV. Thus, our study not only explains the reason for the structural diversity of dUTPase but also reveals how to predict dUTPase type, which may have implications for the dUTPase family. Finally, we tested two dUTPase inhibitors developed for the Plasmodium falciparum dUTPase against the swine and ASFV enzymes. One of these compounds inhibited the ASFV dUTPase at low micromolar concentrations (Kd = 15.6 µM) and with some selectivity (∼2x) over swine dUTPase. In conclusion, our study expands our understanding of the dUTPase family and may aid in the development of specific ASFV inhibitors.

Role of NUDIX Hydrolases in NAD and ADP-Ribose Metabolism in Mammals.

Proteins of the NUDIX hydrolase (NUDT) superfamily that cleave organic pyrophosphates are found in all classes of organisms, from archaea and bacteria to higher eukaryotes. In mammals, NUDTs exhibit a wide range of functions and are characterized by different substrate specificity and intracellular localization. They control the concentration of various metabolites in the cell, including key regulatory molecules such as nicotinamide adenine dinucleotide (NAD), ADP-ribose, and their derivatives. In this review, we discuss the role of NUDT proteins in the metabolism of NAD and ADP-ribose in human and animal cells.

Plasticity, ligand conformation and enzyme action of Mycobacterium smegmatis MutT1.

Mycobacterium smegmatis MutT1 (MsMutT1) is a sanitation enzyme made up of an N-terminal Nudix hydrolase domain and a C-terminal domain resembling a histidine phosphatase. It has been established that the action of MutT1 on 8-oxo-dGTP, 8-oxo-GTP and diadenosine polyphosphates is modulated by intermolecular interactions. In order to further explore this and to elucidate the structural basis of its differential action on 8-oxo-NTPs and unsubstituted NTPs, the crystal structures of complexes of MsMutT1 with 8-oxo-dGTP, GMPPNP and GMPPCP have been determined. Replacement soaking was used in order to ensure that the complexes were isomorphous to one another. Analysis of the structural data led to the elucidation of a relationship between the arrangements of molecules observed in the crystals, molecular plasticity and the action of the enzyme on nucleotides. The dominant mode of arrangement involving a head-to-tail sequence predominantly leads to the generation of NDPs. The other mode of packing arrangement appears to preferentially generate NMPs. This work also provides interesting insights into the dependence of enzyme action on the conformation of the ligand. The possibility of modulating the enzyme action through differences in intermolecular interactions and ligand conformations makes MsMutT1 a versatile enzyme.

Crystal structures of human ENPP1 in apo and bound forms.

Cancer is one of the leading causes of mortality in humans, and recent work has focused on the area of immuno-oncology, in which the immune system is used to specifically target cancerous cells. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is an emerging therapeutic target in human cancers owing to its role in degrading cyclic GMP-AMP (cGAMP), an agonist of the stimulator of interferon genes (STING). The available structures of ENPP1 are of the mouse enzyme, and no structures are available with anything other than native nucleotides. Here, the first X-ray crystal structures of the human ENPP1 enzyme in an apo form, with bound nucleotides and with two known inhibitors are presented. The availability of these structures and a robust crystallization system will allow the development of structure-based drug-design campaigns against this attractive cancer therapeutic target.

Development of a chemical probe against NUDT15.

The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides, but was later shown to hydrolyze the active thiopurine metabolites, 6-thio-(d)GTP, thereby dictating the clinical response of this standard-of-care treatment for leukemia and inflammatory diseases. Nonetheless, its physiological roles remain elusive. Here, we sought to develop small-molecule NUDT15 inhibitors to elucidate its biological functions and potentially to improve NUDT15-dependent chemotherapeutics. Lead compound TH1760 demonstrated low-nanomolar biochemical potency through direct and specific binding into the NUDT15 catalytic pocket and engaged cellular NUDT15 in the low-micromolar range. We also employed thiopurine potentiation as a proxy functional readout and demonstrated that TH1760 sensitized cells to 6-thioguanine through enhanced accumulation of 6-thio-(d)GTP in nucleic acids. A biochemically validated, inactive structural analog, TH7285, confirmed that increased thiopurine toxicity takes place via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues.