Crystal structure of human cytosolic 5'-nucleotidase II: insights into allosteric regulation and substrate recognition

Cytosolic 5'-nucleotidase II catalyzes the dephosphorylation of 6-hydroxypurine nucleoside 5'-monophosphates and regulates the IMP and GMP pools within the cell. It possesses phosphotransferase activity and thereby also catalyzes the reverse reaction. Both reactions are allosterically activated by adenine-based nucleotides and 2,3-bisphosphoglycerate. We have solved structures of cytosolic 5'-nucleotidase II as native protein (2.2 Angstrom) and in complex with adenosine (1.5 Angstrom) and beryllium trifluoride (2.15 Angstrom) The tetrameric enzyme is structurally similar to enzymes of the haloacid dehalogenase (HAD) superfamily, including mitochondrial 5'(3')-deoxyribonucleotidase and cytosolic 5'-nucleotidase III but possesses additional regulatory regions that contain two allosteric effector sites. At effector site 1 located near a subunit interface we modeled diadenosine tetraphosphate with one adenosine moiety in each subunit. This efficiently glues the tetramer subunits together in pairs. The model shows why diadenosine tetraphosphate but not diadenosine triphosphate activates the enzyme and supports a role for cN-II during apoptosis when the level of diadenosine tetraphosphate increases. We have also modeled 2,3-bisphosphoglycerate in effector site 1 using one phosphate site from each subunit. By comparing the structure of cytosolic 5'-nucleotidase II with that of mitochondrial 5'(3')-deoxyribonucleotidase in complex with dGMP, we identified residues involved in substrate recognition.     

Structural basis for the allosteric regulation and substrate recognition of human cytosolic 5'-nucleotidase II

Cytosolic 5′-nucleotidase II (cN-II) catalyzes the dephosphorylation of 6-hydroxypurine nucleoside 5′-monophosphates and participates in the regulation of purine nucleotide pools within the cell. It interferes with the phosphorylation-dependent activation of nucleoside analogues used in the treatment of cancer and viral diseases. It is allosterically activated by a number of phosphate-containing cellular metabolites such as ATP, diadenosine polyphosphates, and 2,3-bisphosphoglycerate, which couple its activity with the metabolic state of the cell. We present seven high-resolution structures of human cN-II, including a ligand-free form and complexes with various substrates and effectors. These structures reveal the structural basis for the allosteric activation of cN-II, uncovering a mechanism where an effector-induced disorder-to-order transition generates rearrangements within the catalytic site and the subsequent coordination of the catalytically essential magnesium. Central to the activation is the large transition of the catalytically essential Asp356. This study also provides the structural basis for the substrate specificity of cN-II, where Arg202, Asp206, and Phe157 seem to be important residues for purine/pyrimidine selectivity. These structures provide a comprehensive structural basis for the design of cN-II inhibitors. They also contribute to the understanding of how the nucleotide salvage pathway is regulated at a molecular level.     

Nucleotide binding to nucleoside diphosphate kinases: X-ray structure of human NDPK-A in complex with ADP and comparison to protein kinases

NDPK-A, product of the nm23-H1 gene, is one of the two major isoforms of human nucleoside diphosphate kinase. We analyzed the binding of its nucleotide substrates by fluorometric methods. The binding of nucleoside triphosphate (NTP) substrates was detected by following changes of the intrinsic fluorescence of the H118G/F60W variant, a mutant protein engineered for that purpose. Nucleoside diphosphate (NDP) substrate binding was measured by competition with a fluorescent derivative of ADP, following the fluorescence anisotropy of the derivative. We also determined an X-ray structure at 2.0A resolution of the variant NDPK-A in complex with ADP, Ca(2+) and inorganic phosphate, products of ATP hydrolysis. We compared the conformation of the bound nucleotide seen in this complex and the interactions it makes with the protein, with those of the nucleotide substrates, substrate analogues or inhibitors present in other NDP kinase structures. We also compared NDP kinase-bound nucleotides to ATP bound to protein kinases, and showed that the nucleoside monophosphate moieties have nearly identical conformations in spite of the very different protein environments. However, the beta and gamma-phosphate groups are differently positioned and oriented in the two types of kinases, and they bind metal ions with opposite chiralities. Thus, it should be possible to design nucleotide analogues that are good substrates of one type of kinase, and poor substrates or inhibitors of the other kind.     

Kinetic properties of human thymidylate synthase,an anticancer drug target

We have determined the kinetic parameters of human recombinant thymidylate synthase (hrTS) with its natural substrate, dUMP, and E-5-(2-bromovinyl)-2(')-deoxyuridine monophosphate (BVdUMP), a nucleotide derivative believed to be the active species of the novel anticancer drug NB1011. NB1011 is activated by hrTS and is selectively toxic to high thymidylate synthase expressing tumor cells. BVdUMP undergoes hrTS-catalyzed thiol-dependent transformation. dUMP and BVdUMP act as competitive hrTS substrates. The natural folate cofactor, CH(2)-THF, inhibits the TS-catalyzed reaction with BVdUMP. We suggest that lower folate levels found in tumor cells favor TS-catalyzed BVdUMP transformation, which, in addition to higher levels of TS expression in tumor cells, contributes to the favorable therapeutic index of the drug NB1011.     

Structural basis for substrate specificity of the human mitochondrial deoxyribonucleotidase

The human mitochondrial deoxyribonucleotidase catalyzes the dephosphorylation of thymidine and deoxyuridine monophosphates and participates in the regulation of the dTTP pool in mitochondria. We present seven structures of the inactive D41N variant of this enzyme in complex with thymidine 3'-monophosphate, thymidine 5'-monophosphate, deoxyuridine 5'-monophosphate, uridine 5'-monophosphate, deoxyguanosine 5'-monophosphate, uridine 2'-monophosphate, and the 5'-monophosphate of the nucleoside analog 3'-deoxy 2'3'-didehydrothymidine, and we draw conclusions about the substrate specificity based on comparisons with enzyme activities. We show that the enzyme's specificity for the deoxyribo form of nucleoside 5'-monophosphates is due to Ile-133, Phe-49, and Phe-102, which surround the 2' position of the sugar and cause an energetically unfavorable environment for the 2'-hydroxyl group of ribonucleoside 5'-monophosphates. The close binding of the 3'-hydroxyl group of nucleoside 5'-monophosphates to the enzyme indicates that nucleoside analog drugs that are substituted with a bulky group at this position will not be good substrates for this enzyme.     

Novel nucleotide analogues as potential substrates for TMPK, a key enzyme in the metabolism of AZT

Novel cyclic and acyclic analogues of dTMP and AZTMP were synthesized from the corresponding cycloSal-phosphotriesters. This method yielded the nucleotides in good yields with a simple work-up. Investigation of the substrate properties of the modified nucleotides towards TmpK showed, that they are very poor substrates for this key enzyme in the bioactivation of AZT.     

Hepatitis C Virus Non-Structural Protein 3 Interacts with Cytosolic 5′(3′)-Deoxyribonucleotidase and Partially Inhibits Its Activity

Infection with hepatitis C virus (HCV) is etiologically involved in liver cirrhosis, hepatocellular carcinoma and B-cell lymphomas. It has been demonstrated previously that HCV non-structural protein 3 (NS3) is involved in cell transformation. In this study, a yeast two-hybrid screening experiment was conducted to identify cellular proteins interacting with HCV NS3 protein. Cytosolic 5′(3′)-deoxyribonucleotidase (cdN, dNT-1) was found to interact with HCV NS3 protein. Binding domains of HCV NS3 and cellular cdN proteins were also determined using the yeast two-hybrid system. Interactions between HCV NS3 and cdN proteins were further demonstrated by co-immunoprecipitation and confocal analysis in cultured cells. The cellular cdN activity was partially repressed by NS3 protein in both the transiently-transfected and the stably-transfected systems. Furthermore, HCV partially repressed the cdN activity while had no effect on its protein expression in the systems of HCV sub-genomic replicons and infectious HCV virions. Deoxyribonucleotidases are present in most mammalian cells and involve in the regulation of intracellular deoxyribonucleotides pools by substrate cycles. Control of DNA precursor concentration is essential for the maintenance of genetic stability. Reduction of cdN activity would result in the imbalance of DNA precursor concentrations. Thus, our results suggested that HCV partially reduced the cdN activity via its NS3 protein and this may in turn cause diseases.     

Human erythrocytic purine nucleoside phosphorylase: reaction with sugar-modified nucleoside substrates

The kinetic parameters (Km and Vmax) of sugar-modified analogues of inosine and guanosine have been determined with human erythrocytic purine nucleoside phosphorylase (PNP). Steric alterations at the 2' and 3' positions greatly lessened or abolished substrate activity. However, the 5'-deoxy- and 2',5'-dideoxy-beta-D-ribofuranosyl and the alpha-L-lyxosyl analogues were good substrates, indicating that the 5'-hydroxyl and the orientation of the 5'-hydroxy-methyl group are not important for binding. The sugar phosphate analogue, 5-deoxyribose 1-phosphate, was synthesized from 5'-deoxyinosine with immobilized PNP, and its presence was verified by using it in the enzymic synthesis of 5'-deoxyguanosine. The adenosine versions of the 5'-modified analogues were also found to react with adenosine deaminase, albeit at less than 1% of Vmax.     

Characterization of the 3' --> 5' exonuclease activity found in human nucleoside diphosphate kinase 1 (NDK1) and several of its homologues

Nucleoside diphosphate kinases (NDKs), an evolutionarily conserved family of proteins, synthesize nucleoside triphosphates from nucleoside diphosphates and ATP. Here, we have characterized the kinase activity and DNA processing functions of eight human proteins that contain at least one domain homologous to Escherichia coli NDK. Not all human proteins with NDK-like domains exhibited NDK activity when expressed as recombinant proteins in E. coli. Human NDK1 (NM23-H1) has been reported to have 3' --> 5' exonuclease activity. In addition to human NDK1, we also find that human NDK5, NDK7, and NDK8 contain 3' --> 5' exonuclease activity. Site-directed mutagenesis, competition assays between wild-type and mutant NDK proteins, and NMR studies confirmed that the DNA-binding and 3' --> 5' exonuclease activity of human NDK1 is an intrinsic activity of the protein. Using double-stranded DNA substrates containing modified bases, human NDK1 efficiently excised nucleotides from the single-strand break produced by APE1 or Nth1. When human cells were treated with various DNA-damaging agents, human NDK1 translocated from the cytoplasm to the nucleus. These results suggest that, in addition to maintenance of nucleotide pool balance, the human NDK-like proteins may have previously unrecognized roles in DNA nucleolytic processing.     

NMR study of dideoxynucleotides with anti-human immunodeficiency virus (HIV) activity

The molecular structures of 3′-azido-2′,3′-dideoxyribosylthymine 5′-triphosphate (AZTTP), 2′,3′-dideoxyribosylinosine 5′-triphosphate (ddITP), 3′-azido-2′,3′-dideoxyribosylthymine 5′-monophosphate (AZTMP) and 2′,3′-dideoxyribosyladenine 5′-monophosphate (ddAMP) have been studied by NMR to understand their anti-HIV activity. For ddAMP and ddITP, conformations are almost identical with their nucleoside analogues with sugar ring pucker equilibriating between C3′-endo (∼75%) and C2′-endo (∼25%). AZTMP and AZTTP on the other hand show significant variations in the conformational behaviour compared with 3′-azido-2′,3′-dideoxyribo-sylthymine (AZT). The sugar rings for these nucleotides have a much larger population of C2′-endo (∼75%) conformers, like those observed for natural 2′-deoxynucleosides and nucleotides. The major conformers around C5′-O5′, C4′-C5′ and the glycosidic bonds are the β^t, γ⁺ and anti, respectively.     

Fluorescence energy transfer studies of human deoxycytidine kinase: role of cysteine 185 in the conformational changes that occur upon substrate binding

Human deoxycytidine kinase (dCK) phosphorylates both pyrimidine and purine deoxynucleosides, including numerous nucleoside analogue prodrugs. Energy transfer studies of transfer between Trp residues of dCK and the fluorescent probe N-(1-pyrene)maleimide (PM), which specifically labels Cys residues in proteins, were performed. Two of the six Cys residues in dCK were labeled, yielding a protein that was functionally active. We determined the average distances between PM-labeled Cys residues and Trp residues in dCK in the absence and presence of various pyrimidine and purine nucleoside analogues with the Trp residues as energy donors and PM-labeled Cys residues as acceptors. The transfer efficiency was determined from donor intensity quenching and the F?rster distance R(0) at which the efficiency of energy transfer is 50%, which was 19.90 A for dCK-PM. The average distance R between the Trp residues and the labeled Cys residues in dCK-PM was 18.50 A, and once substrates bound, this distance was reduced, demonstrating conformational changes. Several of the Cys residues of dCK were mutated to Ala, and the properties of the purified mutant proteins were studied. PM labeled a single Cys residue in Cys-185-Ala dCK, suggesting that one of the two Cys residues labeled in wild-type dCK was Cys 185. The distance between the single PM-labeled Cys residue and the Trp residues in Cys-185-Ala dCK was 20.75 A. Binding of nucleosides had no effect on the pyrene fluorescence of Cys-185-Ala dCK, indicating that the conformational changes observed upon substrate binding to wild-type dCK-PM involved the "lid region" of which Cys 185 is a part. The substrate specificity of Cys-185-Ala dCK was altered in that dAdo and UTP were better substrates for the mutant than for the wild-type enzyme.     

Novel Nucleotide Analogues as Potential Substrates for TMPK,a Key Enzyme in the Metabolism of AZT

Abstract

Novel cyclic and acyclic analogues of dTMP and AZTMP were synthesized from the corresponding cycloSal-phosphotriesters. This method yielded the nucleotides in good yields with a simple work-up. Investigation of the substrate properties of the modified nucleotides towards TmpK showed, that they are very poor substrates for this key enzyme in the bioactivation of AZT.     

A simple direct assay of 3',5'-cyclic nucleotide phosphodiesterase activity based on the use of polyacrylamide-bononate affinity gel chromatography.

A rapid, simple, and direct assay for 3',5'-cyclic nucleotide phospho-diesterase activity is based on the effective separation of cyclic AMP, cyclic GMP or cyclic CMP from their corresponding 5'-nucleotides and nucleosides by chromatography on a polyacrylamide-boronate gel. The affinity of the boronate residue for cis-diols results in the retention of 5'nucleotides and nucleosides while 3',5'-cyclic nucleotides are not retained. The coelution of all 5'-nucleotides and nucleosides allows for the accurate assessment of phosphodiesterase activity in preparations contaminated by other purine metabolizing enzymes such as 5'-nucleotidases and nucleotide and nucleoside deaminases. Phosphodiesterase activity assayed by this means yields linear reaction kinetics with respect to time and amount of enzyme protein. Low blank values obtained allow for detection of as little as 2-3% conversion of substrate to product.     

Properties of 7-alkylguanosines as substrates of purine nucleoside phosphorylase

A series of 7-alkyl analogues of guanosine was prepared by alkylation of 5'-GMP, and enzymatic dephosphorylation of the products to the corresponding nucleosides. The latter were all excellent, as well as fluorescent, substrates of calf spleen nucleoside phosphorylase. Kinetic parameters demonstrated that the purine ring N(7) is not a binding site for the enzyme.     

Nucleoside hydrolase from Leishmania major. Cloning, expression, catalytic properties, transition state inhibitors, and the 2.5-? crystal structure.

Protozoan parasites lack the pathway of the de novo synthesis of purines and depend on host-derived nucleosides and nucleotides to salvage purines for DNA and RNA synthesis. Nucleoside hydrolase is a central enzyme in the purine salvage pathway and represents a prime target for the development of anti-parasitic drugs. The full-length cDNA for nucleoside hydrolase from Leishmania major was cloned and sequence analysis revealed that the L. major nucleoside hydrolase shares 78% sequence identity with the nonspecific nucleoside hydrolase from Crithidia fasciculata. The L. major enzyme was overexpressed in Escherichia coli and purified to over 95% homogeneity. The L. major nucleoside hydrolase was identified as a nonspecific nucleoside hydrolase since it demonstrates the characteristics: 1) efficient utilization of p-nitrophenyl beta-D-ribofuranoside as a substrate; 2) recognition of both inosine and uridine nucleosides as favored substrates; and 3) significant activity with all of the naturally occurring purine and pyrimidine nucleosides. The crystal structure of the L. major nucleoside hydrolase revealed a bound Ca(2+) ion in the active site with five oxygen ligands from Asp-10, Asp-15 (bidentate), Thr-126 (carbonyl), and Asp-241. The structure is similar to the C. fasciculata IU-nucleoside hydrolase apoenzyme. Despite the similarities, the catalytic specificities differ substantially. Relative values of k(cat) for the L. major enzyme with inosine, adenosine, guanosine, uridine, and cytidine as substrates are 100, 0.5, 0.5, 27 and 0.3; while those for the enzyme from C. fasciculata are 100, 15, 14, 510, and 36 for the same substrates. Iminoribitol analogues of the transition state are nanomolar inhibitors. The results provide new information for purine and pyrimidine salvage pathways in Leishmania.     

Design of nucleoside, oligonucleotide and polynucleotide analogues as antiviral agents

Several approaches can be envisaged in the design of nucleoside and oligo- or polynucleotide analogues with selective antiviral activity: (i) deoxythymidine (dThd) or deoxycytidine (dCyd) analogues which are specifically recognized as substrate by the virus-induced dThd-dCyd kinase; (ii) adenosine analogues which impair transmethylation reactions (or polyamine biosynthesis), by virtue of an inhibition of S-adenosylhomocysteine hydrolase; (iii) (2'-5')-oligonucleotide analogues derived from pppA(2'p5'A)2, an important intermediate in the antiviral action of interferon; (iv) oligo(deoxy)nucleotides that are complementary to a well-defined nucleotide sequence of the viral genome; (v) single-stranded homopolynucleotides that act as antitemplates for virus-associated RNA or DNA polymerases; and (vi) double-stranded homopolynucleotides that may be pursued for their interferon-inducing potentials.     

Nucleoside triphosphate specificity of tubulin

We have determined the binding affinity for binding of the four purine nucleoside triphosphates GTP, ITP, XTP, and ATP to E-site nucleotide- and nucleoside diphosphate kinase-depleted tubulin. The relative binding affinities are 3000 for GTP, 10 for ITP, 2 for XTP, and 1 for ATP. Thus, the 2-exocyclic amino group in GTP is important in determining the nucleotide specificity of tubulin and may interact with a hydrogen bond acceptor group in the protein. The 6-oxo group also makes a contribution to the high affinity for GTP. NMR ROESY experiments indicate that the four nucleotides have different average conformations in solution. ATP and XTP are characterized by a high anti conformation, ITP by a medium anti conformation, and GTP by a low anti conformation. Possibly, the preferred solution conformation contributes to the differences in affinities. When the tubulin E-site is saturated with nucleotide, there appears to be little difference in the ability of the four nucleotides to stimulate assembly. The critical protein concentration is essentially identical in reactions using the four nucleotides. All four of the nucleotides were hydrolyzed during the assembly reaction, and the NDPs were incorporated into the microtubule. We also examined the binding of two gamma-phosphoryl-modified GTP photoaffinity analogues, p(3)-1, 4-azidoanilido-GTP and p(3)-1,3-acetylanilido-GTP. These analogues are inhibitors of the assembly reaction and bind to tubulin with affinities that are 15- and 50-fold lower, respectively, than the affinty for GTP. The affinity of GTP is less sensitive to substitutions at the gamma-phosphoryl position that to changes in the purine ring.     

Interactions of Escherichia coli primary replicative helicase DnaB protein with nucleotide cofactors.

Interactions between the Escherichia coli primary replicative helicase DnaB protein and nucleotide cofactors have been studied using several fluorescent nucleotide analogs and unmodified nucleotides. The thermodynamically rigorous fluorescent titration technique has been used to obtain true binding isotherms, independently of the assumptions of any relationships between the observed quenching of protein fluorescence and the degree of nucleotide binding. Fluorescence titrations using several MANT derivatives of nucleoside diphosphates (MANT-ADP, 3',2'-O-(N-methylantraniloyl)adenosine-5'-diphosphate; MANT-GDP, 3',2'-O(N-methylantraniloyl)guanosine-5'-diphosphate; MANT-CDP, 3',2'-O-(N-methylantraniloyl)cytidine-5'-diphosphate; MANT-UDP, 3',2'-O-(N-methylantraniloyl)uridine-5'-diphosphate) have shown that the DnaB helicase has a preference for purine nucleotides. Binding of all modified nucleotides is characterized by similar negative cooperativity, indicating that negative cooperative interactions are base-independent. Thermodynamic parameters for the interactions of the unmodified nucleotides (ADP, GDP, CDP, and UDP) and inorganic phosphate (P(i)) have been obtained by using the competition titration approach. To analyze multiple ligand binding to a finite circular lattice, for a general case in which each lattice binding site can exist in different multiple states, we developed a matrix method approach to derive analytical expressions for the partition function and the average degree of binding for such cases. Application of the theory to competition titrations has allowed us to extract the intrinsic binding constants and cooperativity parameters for all unmodified ligands. This is the first quantitative estimate of affinities and the mechanisms of binding of different unmodified nucleotides and inorganic phosphate for a hexameric helicase. The intrinsic affinities of all of the studied ATP analogs are lower than the intrinsic affinities of the corresponding ADP analogs. The implications of these results for the mechanism of helicase action are discussed.