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Purified nicotinamide nucleotide transhydrogenase from beef heart was investigated with respect to labeling and subsequent sequence analysis of a nicotinamide nucleotide-binding site. A photo-activated azide derivative, 8-azidoadenosine 5'-monophosphate, was used as an active-site-directed photoaffinity label, which was shown to be specific for the NAD(H)-binding site in the dark. Light-activated incorporation of the label in transhydrogenase was accompanied by an inactivation, which approached 100% at the incorporation of about 1 mol label/mol transhydrogenase monomer. As expected from the assumed site-specificity of the label. NADH prevented both labeling and inactivation to some extent. However, NADPH also prevented labeling and inactivation marginally. The oxidized substrates NAD+ and NADP+ were inhibitory by themselves under these conditions, and the substrate analogs 5'-AMP and 2'-AMP were also poor protectors. The NAD(H)-site specificity of the azido compound was thus largely lost upon illumination and covalent modification. Radioactive labeling of transhydrogenase with 8-azido-[2-3H]-adenosine 5'-monophosphate followed by protease digestion, isolation of labeled peptides and amino-acid sequence analysis showed that Tyr 1006 in the sequence 1001-1027 close to the C-terminus was labeled. This sequence shows homologies with nucleotide-binding sequences in, e.g., F1-ATPase. On the basis of sequence homologies with other NAD(P)-dependent enzymes it is proposed that transhydrogenase contains 4 nucleotide-binding sites, of which 2 constitute the adenine nucleotide-binding domains of the catalytic sites for NAD(H) and NADP(H) close to the N- and C-terminals, respectively. Each of these domains has an additional vicinal nucleotide-binding sequence which may constitute a non-catalytic nucleotide-binding site or the nicotinamide nucleotide-binding domain of the catalytic site. The present results indicate that 8-azidoadenosine 5'-monophosphate is kinetically specific for the catalytic NAD(H)-binding site, but reacts covalently with Tyr 1006 of the putative non-catalytic site or nicotinamide nucleotide-binding domain formed by the 1001-1027 amino acid sequence of the catalytic NADP(H)-binding site. Interactions between the catalytic NAD(H) and NADP(H) binding sites, and the assumed non-catalytic sites, may be facilitated by a ligand-triggered formation of a narrow pocket, which normally allows an efficient hydride ion transfer between the natural substrates.
The Fourier transform NMR pulsed-gradient spin-echo self-diffusion technique was used for studies of nucleotides (AMP, CMP, GMP and UMP) with Li+ or Cs+ added, in 2H2O. 1H-, 7Li- and 133Cs-NMR-based self-diffusion data on the constituents provide a picture of both the degree of ion binding to nucleotides and the self-association of nucleotides in aqueous solution. Self-diffusion coefficients were investigated in a concentration range up to 0.3 molal nucleotide in 2H2O, while keeping the metal ion concentration of Li+ or Cs+ at twice the nucleotide concentration throughout the investigations. The self-association studies reveal that the aggregation constants of the Li salts differ only slightly from the corresponding constants for the disodium salts of the mononucleotides. Within a two-site bound-free model for the counterions and a cooperative indefinite aggregation model for the nucleotides one finds that the degree of ion binding for all these nucleotide systems remains approximately constant, in spite of increasing aggregate concentration. This corresponds to the well-known polyelectrolyte ion condensation behaviour, indicating that large aggregates are formed, supporting previous findings by the present authors on the aggregation behaviour of nucleotides. An observed large effect on the 17O relaxation of water in nucleotide systems can only be reconciled with the presence of relatively large aggregates in solution.
Both ATP and a bivalent nucleotide-bound metal activator, normally Mg2+, are required for nitrogenase activity. EPR and ESEEM (electron spin-echo envelope modulation) measurements have been carried out on adenosine nucleotides in which the Mg2+ ion that is usually bound is replaced by Mn2+ in the presence of Kp2 (nitrogenase Fe-protein from Klebsiella pneumoniae). The Mn2+ zero-field splitting parameters have been determined from the EPR-spectrum to be |D|=0.0125 cm(-1) with a rhombicity lambda=E/D=0.31 by direct diagonalization of the complete spin Hamiltonian. ESEEM spectra of the Fe-protein with MnADP and MnATP both show an ESEEM line pair with one signal component at about 3.6 MHz and a relatively broad resonance at 8 MHz originating from a superhyperfine coupling to a 31P nuclear spin from one or more directly co-ordinated phospho group(s) of the nucleotide. A pronounced resonance overlapping the low-frequency component of the (31)P-signal at about 3.5 MHz is attributed to an interaction of Mn2+ with univalent 23Na nuclei. ESEEM lines at frequencies <3.5 MHz have been ascribed to interactions with 14N nuclei. Differences in the 14N features that depend on the type of nucleotide are consistent with substantial conformational rearrangements at the nucleotide-binding site upon hydrolysis. In addition, four-pulse HYSCORE (hyperfine sublevel correlation spectroscopy) experiments not only confirm the three-pulse ESEEM results, but also achieve significantly better spectral deconvolution, especially of the 31P-couplings, and demonstrate that the nucleotide is at least a unidentate ligand of Mn2+. Moreover it was also possible to identify peaks from an 14N interaction more clearly; these most probably arise from outer-sphere interactions with nitrogen atom(s) of non-co-ordinated residues which are affected by conformational rearrangements upon nucleotide hydrolysis. In addition, different redox states of the [4Fe-4S] cluster of the Fe-protein show disparate conformations of the metal-nucleotide co-ordination environment, demonstrating that also the cluster site communicates with the nucleotide binding site.
G proteins act as molecular switches in which information flow depends on whether the bound nucleotide is GDP ("off") or GTP ("on"). We studied the basal and receptor-catalyzed nucleotide exchange rates of site-directed mutants of the alpha subunit of transducin. We identified three amino acid residues (Thr-325, Val-328, and Phe-332) in which mutation resulted in dramatic increases (up to 165-fold) in basal nucleotide exchange rates in addition to enhanced receptor-catalyzed nucleotide exchange rates. These three residues are located on the inward facing surface of the alpha5 helix, which lies between the carboxyl-terminal tail and a loop contacting the nucleotide-binding pocket. Mutation of amino acid residues on the outward facing surface of the same alpha5 helix caused a decrease in receptor-catalyzed nucleotide exchange. We propose that the alpha5 helix comprises a functional microdomain in G proteins that affects basal nucleotide release rates and mediates receptor-catalyzed nucleotide exchange at a distance from the nucleotide-binding pocket.
The complete sequence of the genome of the Japanese encephalitis virus (JEV) Ling strain isolated from the brain of a patient in Taiwan in 1965 was cloned by using the reverse transcription-polymerase chain reaction method. Seven overlapping cDNA clones that span the entire virus genome were isolated and sequenced to determine the complete nucleotide sequence, which is 10,951 nucleotides in length. As reported for three other JEV strains (Beijing-1, SA-14, and JaOArS982), the Ling strain contains 95 nucleotides in the 5' nontranslated region (NTR), followed by a single open reading frame of 10,296 nucleotides. However, the length of the 3' NTR of JEV Ling is 560 nucleotides, 25 nucleotides shorter than that of other JEV strains sequenced to date. Comparison of nucleotide and amino acid sequences among these four JEV strains showed that nucleotide (amino acid) sequence divergence in the translated region varied from 1.25% to 3.27% (0.49-1.63%). The nucleotide (amino acid) divergences between the Ling and Beijing-1 strains were 1.25% (0.87%) and between the SA-14 and JaOArS982 strains were 1.42% (0.49%). These values are lower than those found between the Ling and SA-14 [2.44% (1.02%)] or the Ling and JaOArS982 strains [2.84% (0.93%)], as well as those between Beijing-1 and SA-14 [3.14% (1.60%)] or Beijing-1 and JaOArS982 [3.27% (1.63%)] strains. Sequence comparisons of subregions of the genomes i.e., structural genes, nonstructural genes, or individual genes, showed divergence similar to that obtained by comparing the entire sequence. It is likely that the JEV sequence divergence between two human isolates or between two mosquito isolates is lower than that between a human isolate and a mosquito isolate.
Extracellular nucleotides play many biological roles, including intercellular communication and modulation of nucleotide receptor signaling, and are dependent on the phosphorylation state of the nucleotide. Regulation of nucleotide phosphorylation is necessary, and a specialized class of enzymes, nucleotide pyrophosphatases/phosphodiesterases (E-NPPs), has been identified in mammals to perform this function. Although the E-NPP class is conserved among complex eukaryotes, this system has not yet been identified in Saccharomyces cerevisiae. Using genetic and biochemical experiments, we show that two orthologs of the E-NPP family, referred to as Npp1p and Npp2p, exist in budding yeast and can perform nucleotide phosphate hydrolysis. This activity is enhanced during phosphate starvation, where hydrolyzed phosphates can be imported from extracellular sources and utilized to overcome phosphate starvation through the activity of the Pho5p acid phosphatase. The added compensatory effect by Pho5p is also a newly established role for Pho5p. This study demonstrates that extracellular nucleotide phosphate metabolism appears to be controlled by at least two independent regulatory mechanisms, uniting phosphate starvation with extracellular nucleotide regulation.
RecA protein plays a crucial role in homologous recombination and repair of DNA. Central to all activities of RecA is its binding to Mg(+2)-ATP. The active form of the protein is a helical nucleoprotein filament containing the nucleotide cofactor and single-stranded DNA. The stability and structure of the helical nucleoprotein filament formed by RecA are modulated by nucleotide cofactors. Here we report crystal structures of a MtRecA-ADP complex, complexes with ATPgammaS in the presence and absence of magnesium as well as a complex with dATP and Mg+2. Comparison with the recently solved crystal structures of the apo form as well as a complex with ADP-AlF4 confirms an expansion of the P-loop region in MtRecA, compared to its homologue in Escherichia coli, correlating with the reduced affinity of MtRecA for ATP. The ligand bound structures reveal subtle variations in nucleotide conformations among different nucleotides that serve in maintaining the network of interactions crucial for nucleotide binding. The nucleotide binding site itself, however, remains relatively unchanged. The analysis also reveals that ATPgammaS rather than ADP-AlF4 is structurally a better mimic of ATP. From among the complexed structures, a definition for the two DNA-binding loops L1 and L2 has clearly emerged for the first time and provides a basis to understand DNA binding by RecA. The structural information obtained from these complexes correlates well with the extensive biochemical data on mutants available in the literature, contributing to an understanding of the role of individual residues in the nucleotide binding pocket, at the molecular level. Modeling studies on the mutants again point to the relative rigidity of the nucleotide binding site. Comparison with other NTP binding proteins reveals many commonalties in modes of binding by diverse members in the structural family, contributing to our understanding of the structural signature of NTP recognition.
Procedures for the analysis of cellular purine and pyrimidine nucleotides are described. The commonly used perchloric acid and especially the trichloroacetic acid methods for nucleotide extraction interfere with ion-pair high-performance liquid chromatography, but we have developed such a system for the separation and determination of major cellular nucleotides in biological matrices, including tri-, di-, monophosphates, cAMP, cGMP, NAD, NADP, UDP-glucose and UDP-galactose. Compared with perchloric acid extraction, no degradation of the nucleotide standards used was observed with respect to triphosphates and other relatively unstable nucleotides. Cellular nucleotides were extracted by lysing cells in a hypotonic buffer containing an ion-pair reagent (tetrabutylammonium hydrogen-sulphate) to decrease enzymic degradation of nucleotides in combination with ultrafiltration of the cell lysate to remove compounds of higher molecular mass, for example enzymes. This method is a simple and reproducible procedure for investigating nucleotide pools in cells.
ATP binding to the large tumor (T) antigen encoded by the simian virus 40 (SV40) genome plays an essential role in the replication of viral DNA [Fanning, E., and Knippers, R. (1992) Annu. Rev. Biochem. 61, 55-85]. To better explore the functions of T antigen during the replication process, we have studied the interactions of T antigen with fluorescent 3'(2')-O-(2,4,6-trinitrophenyl) (TNP) adenine nucleotide analogues. Binding of TNP-ATP and TNP-ADP was accompanied by an 8-fold fluorescence enhancement and a concomitant blue shift (11 nm) of the maximal emission wavelength; the intrinsic protein tryptophan fluorescence was quenched maximally by 50%. Both signals were utilized to characterize the nucleotide binding activity of T antigen. TNP-ATP and TNP-ADP bound to the ATP binding site with dissociation constants of 0.35 microM and 2.6 microM. TNP substitution enhanced the affinity of ADP for T antigen by approximately 11-fold. The binding stoichiometry was 1 mol of TNP nucleotide per mole of monomer T antigen. The binding of TNP-ATP was more temperature dependent than that of TNP-ADP. The enthalpy change contributed nearly half of the energy for TNP-ATP binding, whereas binding of TNP-ADP was primarily entropy driven. Both TNP-ATP and TNP-ADP were strong inhibitors of the T antigen ATPase activity, confirming the high affinities of the TNP nucleotides for the ATP binding site. Like the parent nucleotides, they also induced T antigen hexamer formation. Using the TNP nucleotides as fluorescent probes, we have measured the affinity of various nucleotides and analogues for T antigen. The results indicate that the nucleotide binding specificity of T antigen was similar to that of the prokaryotic helicases Dna B and Rep, suggesting closely related ATP binding sites in the three DNA helicases.
The removal of tightly bound GDP from the exchangeable nucleotide-binding site of tubulin has been performed with alkaline phosphatase under conditions which essentially retain the assembly properties of the protein. When microtubule protein is treated with alkaline phosphatase, nucleotide is selectively removed from tubulin dimer rather than from MAP (microtubule-associated protein)-containing oligomeric species. Tubulin devoid of E-site (the exchangeable nucleotide-binding site of the tubulin dimer) nucleotide shows enhanced proteolytic susceptibility of the beta-subunit to thermolysin and decreased protein stability, consistent with nucleotide removal causing changes in protein tertiary structure. Pyrophosphate ion (3 mM) is able to promote formation of normal microtubules in the complete absence of GTP by incubation at 37 degrees C either with nucleotide-depleted microtubule protein or with nucleotide-depleted tubulin dimer to which MAPs have been added. The resulting microtubules contain up to 80% of tubulin lacking E-site nucleotide. In addition to its effects on nucleation, pyrophosphate competes weakly with GDP bound at the E-site. It is deduced that binding of pyrophosphate at a vacant E-site can promote microtubule assembly. The minimum structural requirement for ligands to induce tubulin assembly apparently involves charge neutralization at the E-site by bidentate ligation, which stabilizes protein domains in a favourable orientation for promoting the supramolecular protein-protein interactions involved in microtubule formation.
The human multidrug transporter (MDR1 or P-glycoprotein) is an ATP-dependent cellular drug extrusion pump, and its function involves a drug-stimulated, vanadate-inhibited ATPase activity. In the presence of vanadate and MgATP, a nucleotide (ADP) is trapped in MDR1, which alters the drug binding properties of the protein. Here, we demonstrate that the rate of vanadate-dependent nucleotide trapping by MDR1 is significantly stimulated by the transported drug substrates in a concentration-dependent manner closely resembling the drug stimulation of MDR1-ATPase. Non-MDR1 substrates do not modulate, whereas N-ethylmaleimide, a covalent inhibitor of the ATPase activity, eliminates vanadate-dependent nucleotide trapping. A deletion in MDR1 (Delta amino acids 78-97), which alters the substrate stimulation of its ATPase activity, similarly alters the drug dependence of nucleotide trapping. MDR1 variants with mutations of key lysine residues to methionines in the N-terminal or C-terminal nucleotide binding domains (K433M, K1076M, and K433M/K1076M), which bind but do not hydrolyze ATP, do not show nucleotide trapping either with or without the transported drug substrates. These data indicate that vanadate-dependent nucleotide trapping reflects a drug-stimulated partial reaction of ATP hydrolysis by MDR1, which involves the cooperation of the two nucleotide binding domains. The analysis of this drug-dependent partial reaction may significantly help to characterize the substrate recognition and the ATP-dependent transport mechanism of the MDR1 pump protein.
The interaction of nucleotides with pertussis toxin (PT), and their effects on the ability of the toxin to ADP-ribosylate pure Ni, were evaluated. [32P]ATP (10 nM) bound directly to dithiothreitol-activated PT. This binding was competitively inhibited by nucleotides and anions with the following IC50 concentrations in order of decreasing potency: ATP = ATP gamma S (adenosine-5'-O-(3-thiotriphosphate)) = 0.2-0.3 microM, GDP beta S (guanosine-5'-O-(2-thiodiphosphate)) = 2-3 microM, GTP gamma S (guanosine-5'-O-(3-thiotriphosphate)) = 10-15 microM, ADP = 20-25 microM, GTP = 30-40 microM, GMP-P(NH)P (guanyl-5'-yl imidodiphosphate) = 100-150 microM, GDP = 150-200 microM, Pi = SO4(2-) = 20 mM and Cl- = acetate = 30-35 mM. Treatment of PT with ATP, AMP-P(NH)P, GTP, GDP, or GDP beta S, resulted in a stimulated state of NAD+-Ni ADP-ribosyltransferase activity. Addition of ATP, AMP-P(NH)P (adenyl-5'-yl imidodiphosphate), GTP, GDP, and GDP beta S to the ADP-ribosylation reactions resulted in increased rates of ADP-ribosyl-Ni formation. It is concluded that these effects on the nucleotides are due to their action to stimulate the activity of PT. At concentrations of PT between 0.04 and 0.4 microgram/ml, the stimulation of ADP-ribosylation of Ni effected by nucleotides was hysteretic in nature, exhibiting an approximately 25-min long lag when GDP was used as the activating nucleotide. These lags decreased with increasing concentrations of PT, and were abolished by pretreatment of the toxin with GDP or ATP. Preliminary incubation of Ni with GDP had no effect on the lag in its ADP-ribosylation by non-nucleotide treated PT. Addition of divalent cations (Mg2+, Mn2+, and Ca2+) inhibited formation of ADP-ribosyl-Ni, possibly by causing aggregation and denaturation of Ni. This is the first demonstration that both adenine and guanine nucleotides interact directly with PT and act to stimulate its activity to ADP-ribosylate Ni, and that guanine nucleotides do so regardless of whether they are nucleoside di- or triphosphates.
1. The contents of the major purine nucleotides in the isolated non-working perfused rat heart varied systematically during 80min of perfusion. In particular the amounts of ATP, ADP, GTP, cyclic AMP and cyclic GMP in the well-oxygenated myocardium showed changes ranging from 25 to 60% of the mean concentrations. The apparent periodicity was about 30min for some and about 60min for other nucleotides. 2. These data are in contrast with measurements of parameters reflecting heart performance, which remained constant over this period of perfusion. 3. The ATP/ADP ratio, the cyclic AMP content, the GTP content and the GTP/GDP ratio in the tissue bore a constant relationship to one another, and all showed the same temporal variation. 4. Increasing the energy demand on the heart by administration of bovine somatotropin (1mug/ml) tended to damp the variations, and generally lower the content of all the nucleotides. 5. The total extractable adenine nucleotide pool also showed systematic temporal variations of as much as 1.3mumol/g wet wt. of tissue within 10min. 6. These variations could not be accounted for as inter-conversion with adenosine, other purine nucleotides, nucleosides or purine-degradation products either in the tissue or in the perfusion medium. No evidence was found in this preparation of the purine nucleotide oscillations described by Lowenstein and his co-workers [see Tornheim & Lowenstein (1975) J. Biol. Chem.250, 6304-6314]. 7. Further, the pool size increases cannot be satisfactorily explained by either synthesis de novo or the breakdown of any purine macromolecular species in the cell. Thus it is suggested that an unsuspected substantial storage form of purine nucleotide may exist in heart.
The function of the human cystic fibrosis transmembrane conductance regulator (CFTR) protein as a chloride channel or transport regulator involves cellular ATP binding and cleavage. Here we describe that human CFTR expressed in insect (Sf9) cell membranes shows specific, Mg2+-dependent nucleotide occlusion, detected by covalent labeling with 8-azido-[alpha-32P]ATP. Nucleotide occlusion in CFTR requires incubation at 37 degrees C, and the occluded nucleotide can not be removed by repeated washings of the membranes with cold MgATP-containing medium. By using limited tryptic digestion of the labeled CFTR protein we found that the adenine nucleotide occlusion preferentially occurred in the N-terminal nucleotide binding domain (NBD). Addition of the ATPase inhibitor vanadate, which stabilizes an open state of the CFTR chloride channel, produced an increased nucleotide occlusion and resulted in the labeling of both the N-terminal and C-terminal NBDs. Protein modification with N-ethylmaleimide prevented both vanadate-dependent and -independent nucleotide occlusion in CFTR. The pattern of nucleotide occlusion indicates significant differences in the ATP hydrolyzing activities of the two NBDs, which may explain their different roles in the CFTR channel regulation.
We investigated substitution patterns and neighboring-nucleotide effects for 2,576,903 single nucleotide polymorphisms (SNPs) publicly available through the National Center for Biotechnology Information (NCBI). The proportions of substitutions were A/G, 32.77%; C/T, 32.81%; A/C, 8.98%; G/T, 9.06%; A/T, 7.46%; and C/G, 8.92%. The two nucleotides immediately neighboring the variable site showed major deviation from genome-wide and chromosome-specific expectations, although lesser biases extended as far as 200 bp. On the 5' side, the biases for A, C, G, and T were 1.43%, 4.91%, -1.70%, and -4.62%, respectively. These biases were -4.44%, -1.59%, 5.05%, and 0.99%, respectively, on the 3' side. The neighboring-nucleotide patterns for transitions were dominated by the hypermutability effects of CpG dinucleotides. Transitions were more common than transversions, and the probability of a transversion increased with increasing A + T content at the two adjacent sites. Neighboring-nucleotide biases were not consistent among chromosomes, with Chromosomes 19 and 22 standing out as different from the others. These data provide genome-wide information about the effects of neighboring nucleotides on mutational and evolutionary processes giving rise to contemporary patterns of nucleotide occurrence surrounding SNPs.
PyrR is a protein that regulates the expression of genes and operons of pyrimidine nucleotide biosynthesis (pyr genes) in many bacteria. PyrR acts by binding to specific sequences on pyr mRNA and causing transcriptional attenuation when intracellular levels of uridine nucleotides are elevated. PyrR from Bacillus subtilis has been purified and extensively studied. In this work, we describe the purification to homogeneity and characterization of recombinant PyrR from the thermophile Bacillus caldolyticus and the crystal structures of unliganded PyrR and a PyrR-nucleotide complex. The B. caldolyticus pyrR gene was previously shown to restore normal regulation of the B. subtilis pyr operon in a pyrR deletion mutant. Like B. subtilis PyrR, B. caldolyticus PyrR catalyzes the uracil phosphoribosyltransferase reaction but with maximal activity at 60 degrees C. Crystal structures of B. caldolyticus PyrR reveal a dimer similar to the B. subtilis PyrR dimer and, for the first time, binding sites for nucleotides. UMP and GMP, accompanied by Mg2+, bind specifically to PyrR active sites. Nucleotide binding to PyrR is similar to other phosphoribosyltransferases, but Mg2+ binding differs. GMP binding was unexpected. The protein bound specific sequences of pyr RNA 100 to 1,000 times more tightly than B. subtilis PyrR, depending on the RNA tested and the assay method; uridine nucleotides enhanced RNA binding, but guanosine nucleotides antagonized it. The new findings of specific GMP binding and its antagonism of RNA binding suggest cross-regulation of the pyr operon by purines.