Phosphate Groups Of Nucleotides Research Articles

Hydration structure and dynamics of phosphoric acid and its anions-Ultrafast 2D-IR spectroscopy and abinitio molecular dynamics simulations.

The hydration shells of phosphate ions and phosphate groups of nucleotides and phospholipid membranes display markedly different structures and hydrogen-bond strengths. Understanding phosphate hydration requires insight into the spatial arrangements of water molecules around phosphates and in thermally activated structure fluctuations on ultrafast time scales. Femtosecond two-dimensional infrared spectroscopy of phosphate vibrations, particularly asymmetric stretching vibrations between 1000 and 1200cm-1, and abinitio molecular dynamics (AIMD) simulations are combined to map and characterize dynamic local hydration structures and phosphate-water interactions. Phosphoric acid H3PO4 and its anions H2PO4-, HPO42-, and PO43- are studied in aqueous environments of different pH value. The hydration shells of phosphates providing OH donor groups in hydrogen bonds with the first water layer undergo ultrafast structural fluctuations, which induce a pronounced spectral diffusion of vibrational excitations on a sub-300fs time scale. With a decreasing number of phosphate OH groups, the hydration shell becomes more ordered and rigid. The 2D-IR line shapes observed with hydrated PO43- ions display a pronounced inhomogeneous broadening, reflecting a distribution of hydration geometries without fast equilibration. The AIMD simulations allow for an in-depth characterization of the hydration geometries with different numbers of water molecules in the first hydration layer and different correlation functions of the fluctuating electric field that the water environment exerts on the vibrational phosphate oscillators.

Coordination Chemistry of Phosphate Groups in Systems Including Copper(II) Ions, Phosphoethanolamine and Pyrimidine Nucleotides.

The activity of phosphate groups of phosphoethanolamine and pyrimidine nucleotides (thymidine 5-monophosphate, cytidine 5-monophosphate and uridine 5'monophosphate) in the process of complexation metal ions in aqueous solution was studied. Using the potentiometric method with computer calculation of the data and spectroscopic methods such as UV-Vis, EPR, 13C and 31P NMR as well as FT-IR, the overall stability constants of the complexes as well as coordination modes were obtained. At lower pH, copper(II) ions are complexed only by phosphate groups, whereas the endocyclic nitrogen atom of nucleotides has been identified as a negative center interacting with the -NH3+ groups of phosphoethanolamine.

Red cell adenylate kinase deficiency in China: molecular study of 2 new mutations (413G > A, 223dupA)

BackgroundAdenylate kinase (AK) is a monomolecular enzyme widely found in a variety of organisms. It mainly catalyses the reversible transfer of adenosine nucleotide phosphate groups and plays an important role in maintaining energy metabolism. AK deficiency is a rare genetic disorder that is related to haemolytic anaemia. Chronic haemolytic anaemia associated with AK deficiency is a rare condition, and only 14 unrelated families have been reported thus far. Moreover, only 11 mutations have been identified in the AK1 gene, with only 3 cases of psychomotor impairment.Case presentationThe patient was a 3-year-old boy with severe haemolytic anaemia and psychomotor retardation. A molecular study of the patient’s AK gene revealed 2 different mutations: a heterozygous missense mutation in exon 6 (c.413G > A) and a heterozygous frameshift mutation in exon 5 (c.223dupA). Molecular modelling analyses indicated that AK gene inactivation resulted in a lack of AK activity. The patient recovered after regular blood transfusion therapy.ConclusionsAK1 deficiency was diagnosed on the basis of low enzymatic activity and the identification of a mutation in the AK1 gene located on chromosome 9q. Here, we report the first case of moderate red cell AK1 deficiency associated with chronic nonspherocytic haemolytic anaemia (CNSHA) in China. The genetic mutations were confirmed by Sanger sequencing. The variants were classified as pathogenic by bioinformatics tools, such as ACMG/AMP guidelines, Mutation Taster, SIFT, MACP, REVEL and PolyPhen2.2. Based on our evidence and previous literature reports, we speculate that the site of the AK1 gene c.413G > A (p.Arg138His) mutation may be a high-frequency mutation site and the other mutation (c.223dupA) might be related to the neuropathogenicity caused by AK1 deficiency. NGS should be a part of newborn to early childhood screening to diagnose rare and poorly diagnosed genetic diseases as early as possible.

AK2 Promotes the Migration and Invasion of Lung Adenocarcinoma by Activating TGF-β/Smad Pathway In vitro and In vivo.

Adenylate kinase 2 (AK2) is a wide-spread and highly conserved protein kinase whose main function is to catalyze the exchange of nucleotide phosphate groups. In this study, we showed that AK2 regulated tumor cell metastasis in lung adenocarcinoma. Positive expression of AK2 is related to lung adenocarcinoma progression and poor survival of patients. Knockdown or knockout of AK2 inhibited, while overexpression of AK2 promoted, human lung adenocarcinoma cell migration and invasion ability. Differential proteomics results showed that AK2 might be closely related to epithelial-mesenchymal transition (EMT). Further research indicated that AK2 regulated EMT occurrence through the Smad-dependent classical signaling pathways as measured by western blot and qPCR assays. Additionally, in vivo experiments showed that AK2-knockout in human lung tumor cells reduced their EMT-like features and formed fewer metastatic nodules both in liver and in lung tissues. In conclusion, we uncover a cancer metastasis-promoting role for AK2 and provide a rationale for targeting AK2 as a potential therapeutic approach for lung cancer.

Investigation of the inhibited biotoxicity of heavy metals towards 5- formylcytosine in rice by hydrochar based on photoelectrochemical biosensor

A photoelectrochemical (PEC) biosensor was constructed for 5-formylcytosine (5fC) nucleotide detection based on Ag2S@WS2 photoactive material and FeVO4 catalytic signal quenching. After Ag2S@WS2 was modified onto the ITO substrate surface, 5fC recognition reagent of Au@4-amino3hydrazino5mercapto-1,2,4-triazol (Au@AHMT) was further modified through electrostatic adsorption. Afterwards, based on the specific chemical reaction between –NH2 and –CHO, 5fC can be selectively recognized and captured. Subsequently, the nanoenzyme of FeVO4 was recognized based on the specific reaction between the phosphate group of 5fC nucleotide and Fe3+. Under the catalysis of FeVO4, the 4-chloro-1-naphthol in the detection solution can be oxidized to generate a precipitate, which will be retained on the electrode surface to inhibit the PEC signal. The developed method presented a widely dynamic range from 0.1 to 400 nM. The detection limit was 0.062 nM (3σ). This method also showed good detection selectivity, reproducibility and stability. The applicability was verified by investigating 5fC content change in genomic DNA of rice tissues after incubated with heavy metals. Moreover, the inhibited influence of hydrochar towards heavy metals was also assessed.

PF197 A NEW CLASSIFICATION OF ACUTE MYELOID LEUKEMIA BASED ON INTEGRATED GENOMICS AND METABOLOMICS

Background:An expanding body of evidence indicates that targeting cell metabolism is a promising therapeutic strategy in acute myeloid leukemia (AML). Pyrimidine biosynthesis, oxidative phosphorylation, amino acid and arginine metabolism are among the most relevant actionable pathways (Sykes et al. Cell 2016; Molina et al. Nat Med 2018; Jones et al. Cancer Cell 2018; Mussai et al. Blood 2015). However, few studies identified specific metabolic vulnerabilites (Fenouille et al. Nat Med 2017; Gallipoli et al. Blood 2018) due to the lack of a comprehensive characterization of AML cell metabolism.Aims:The study aimed to stratify AML patients based on their metabolic landscape and integrate their genomic and metabolic profiles.Methods:Genomic data of AML patients were obtained by whole exome sequencing (n = 165) or targeted NGS (n = 18). Of them, 119 AML were evaluated for serum metabolites by nuclear magnetic resonance (NMR), along with 145 healthy donors. Intracellular metabolites of AML bone marrow cells (35 CD34+ and 15 CD33+CD14− samples) and controls (21 cord blood CD34+ and 21 peripheral blood CD33+ samples) were analyzed by mass spectrometry (Metabolon).Results:Unsupervised hierarchical clustering of AML based on the intracellular metabolic landscape clearly defined 3 clusters (C, Fig.A). C1 was characterized by high metabolite concentration, C2 by low metabolite levels and C3 by a more complex metabolic profile. By integrating genomic data into the metabolic classification we obtained a significant clusterization (p = 0.029). C1 was enriched for NPM1‐mutated (mut) AML (50%), C2 for cases with altered chromatin/splicing genes or inv(3) (37% and 13%, respectively), C3 for TP53‐mut/aneuploid AML (35%). Moreover, C3 included all core binding factor AML. By training a Gaussian Process classifier on NMR data, we obtained a significant separation of C1 and C3 also at serum level (Fig. B,C). In particular, NPM1‐mut AML displayed abundance of tricarboxylic cycle byproducts, while glutamine and creatinine were increased in NPM1‐wildtype (wt) cases (Fig.D,E). At intracellular level, NPM1‐mut AML were characterized by increased intermediates of purine and pyrimidine metabolism, in line with an active biosynthetic activity. Moreover, unsupervised NMR data analysis identified a clear separation (0:7 accuracy) between TP53‐wt and mut/deleted AML, the latter showing reduced threonine, alanine, glucose, lactate and glutamine serum concentration (Fig.F).At genomic level, 88% of patients carried at least one mutation in a metabolism‐related gene including enzymes (encoded by nuclear or mitchondrial DNA) and metabolic regulators, with lipid metabolism, cellular respiration (IDH1/2 mutations and oxidative phosphorylation with altered UQCRH, FASTKD3, ND and COX genes), metabolism of carbohydrates (e.g. glycosyltransferase GXYLT1, GDP‐mannose biosynthetic process involving PMM2), glucose (e.g. glycolysis‐related genes HK2, HK3, PKLR, BPGM) and nucleotides (e.g. AK2, AK5: nucleotide phosphate group transfer, NME7: synthesis of nucleoside triphosphates) being the pathways most frequently targeted by mutations.Summary/Conclusion:AML is characterized by a number of functional and genomic‐driven metabolic alterations. The integration of genomic and metabolic profiles provided a novel refined AML classification and suggested subtype‐specific metabolic targets, including nucleotide metabolic pathways and bioenergetics in NPM1‐mut and TP53‐mut/del AML, respectively.imageSupported by: EHA research fellowship award, AIRC, FP7‐NGS‐PTL, Fondazione del Monte.

Structural Insights into Substrate Selectivity and Activity of Bacterial Polyphosphate Kinases

Polyphosphate (polyP) kinases are widely conserved enzymes with importance in basic bacterial metabolism and virulence in many pathogens. However, the molecular mechanisms of their substrate specificity and catalysis remain unknown. Here, we present the results of comprehensive biochemical and structural studies of three polyP kinases from different bacteria, which belong to different clusters of the PPK2 class III family. Purified PPK2 proteins catalyzed polyP-dependent phosphorylation of AMP, ADP, GMP, and GDP to corresponding nucleoside diphosphates and triphosphates. Crystal structures of these proteins in complex with substrates, products, Mg2+, and inhibitors revealed the binding sites for the nucleotide and polyP substrates overlapping at the Walker A and B loops. The Walker A loop is involved in the binding of polyP and the Mg2+ ion, whereas the Walker B loop coordinates the nucleotide phosphate groups. Structure-based site-directed mutagenesis of CHU0107 from Cytophaga hutchinsonii demonstrated t...

Computational investigation of proton transfer, pKa shifts and pH-optimum of protein-DNA and protein-RNA complexes.

Protein-nucleic acid interactions play a crucial role in many biological processes. This work investigates the changes of pKa values and protonation states of ionizable groups (including nucleic acid bases) that may occur at protein-nucleic acid binding. Taking advantage of the recently developed pKa calculation tool DelphiPka, we utilize the large protein-nucleic acid interaction database (NPIDB database) to model pKa shifts caused by binding. It has been found that the protein's interfacial basic residues experience favorable electrostatic interactions while the protein acidic residues undergo proton uptake to reduce the energy cost upon the binding. This is in contrast with observations made for protein-protein complexes. In terms of DNA/RNA, both base groups and phosphate groups of nucleotides are found to participate in binding. Some DNA/RNA bases undergo pKa shifts at complex formation, with the binding process tending to suppress charged states of nucleic acid bases. In addition, a weak correlation is found between the pH-optimum of protein-DNA/RNA binding free energy and the pH-optimum of protein folding free energy. Overall, the pH-dependence of protein-nucleic acid binding is not predicted to be as significant as that of protein-protein association. Proteins 2017; 85:282-295. © 2016 Wiley Periodicals, Inc.

High-Throughput Screening for Ligands of the HEPN Domain of Sacsin.

Sacsin is a large protein implicated in the neurodevelopmental and neurodegenerative disease autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), which features the loss of Purkinje neurons in the cerebellum. Although the domain architecture of sacsin suggests that it is a neuronal chaperone assisting in protein quality control, the precise function of sacsin remains elusive. Using fluorescence polarization (FP) assays, we confirmed that the HEPN domain of sacsin binds to nucleotides with low micromolar affinities. FP competition assays with a variety of nucleotides and nucleotide analogs revealed that the binding is primarily mediated by the phosphate groups of nucleotides. A high-throughput screen subsequently identified novel small molecule ligands of HEPN, providing new chemical probes for cell culture studies and drug development. Together, the results are consistent with the HEPN domain contributing to the functional activity of sacsin by binding to nucleotides or other multiply charged anionic compounds in neurons.

Recognition rules for binding of Zn-Cys2His2 transcription factors to operator DNA

The molecules of Zn-finger transcription factors consist of several similar small protein units. We analyzed the crystal structures 46 basic units of 22 complexes of Zn-Cys2His2 family with the fragments of operator DNA. We showed that the recognition of DNA occurs via five protein contacts. The canonical binding positions of the recognizing α-helix were −1, 3, 6, and 7, which make contacts with the tetra-nucleotide sequence ZXYZ of the coding DNA strand; here the canonical binding triplet is underlined. The non-coding DNA strand forms only one contact at α-helix position 2. We have discovered that there is a single highly conservative contact His7α with the phosphate group of nucleotide Z, which precedes each triplet XYZ of the coding DNA chain. This particular contact is invariant for the all Zn-Cys2His2 family with high frequency of occurrence 83%, which we considered as an invariant recognition rule. We have also selected a previously unreported Zn-Cys2His2-Arg subfamily of 21 Zn-finger units bound with DNA triplets, which make two invariant contacts with residues Arg6α and His7α with the coding DNA chain. These contacts show frequency of occurrence 100 and 90%, and are invariant recognition rule. Three other variable protein-DNA contacts are formed mainly with the bases and specify the recognition patterns of individual factor units. The revealed recognition rules are inherent for the Zn-Cys2His2 family and Zn-Cys2His2-Arg subfamily of different taxonomic groups and can distinguish members of these families from any other family of transcription factors.

Structure of Mycobacterium tuberculosis nucleoside diphosphate kinase R80N mutant in complex with citrate.

The crystal structure of the wild-type nucleoside diphosphate kinase from Mycobacterium tuberculosis at 2.6 Å resolution revealed that the intersubunit salt bridge Arg80-Asp93 contributes to the thermal stability of the hexamer (Tm= 76°C). On mutating Asp93 to Asn to break the salt bridge, the thermal stability dramatically decreased by 27.6°C. Here, on mutating Arg80 to Asn, the thermal stability also significantly decreased by 8.0°C. In the X-ray structure of the R80N mutant solved at 1.9 Å resolution the salt bridge was replaced by intersubunit hydrogen bonds that contribute to the thermal stability of the hexamer. A citrate anion from the crystallization buffer was bound at the bottom of the nucleotide-binding site via electrostatic and hydrogen-bonding interactions with six conserved residues involved in nucleotide binding. Structural analysis shows that the citrate is present at the location of the nucleotide phosphate groups.

Structural Characterization of the RLCK Family Member BSK8: A Pseudokinase with an Unprecedented Architecture

Brassinosteroid signaling kinases (BSKs) are plant-specific receptor-like cytoplasmic protein kinases involved in the brassinosteroid signaling pathway. Unlike common protein kinases, they possess a naturally occurring alanine residue at the “gatekeeper” position, as well as other sequence variations. How BSKs activate downstream proteins such as BSU1, as well as the structural consequences of their unusual sequential features, was unclear. We crystallized the catalytic domain of BSK8 and solved its structure by multiple-wavelength anomalous dispersion phasing methods to a resolution of 1.5Å. In addition, a co-crystal structure of BSK8 with 5-adenylyl imidodiphosphate (AMP-PNP) revealed unusual conformational arrangements of the nucleotide phosphate groups and catalytic key motifs, typically not observed for active protein kinases. Sequential analysis and comparisons with known pseudokinase structures suggest that BSKs represent constitutively inactive protein kinases that regulate brassinosteroid signal transfer through an allosteric mechanism.

The substrate specificity of the human ADP/ATP carrier AAC1

The mitochondrial ADP/ATP carrier imports ADP from the cytosol into the mitochondrial matrix for its conversion to ATP by ATP synthase and exports ATP out of the mitochondrion to replenish the eukaryotic cell with chemical energy. Here the substrate specificity of the human mitochondrial ADP/ATP carrier AAC1 was determined by two different approaches. In the first the protein was functionally expressed in Escherichia coli membranes as a fusion protein with maltose binding protein and the effect of excess of unlabeled compounds on the uptake of [(32)P]-ATP was measured. In the second approach the protein was expressed in the cytoplasmic membrane of Lactococcus lactis. The uptake of [(14)C]-ADP in whole cells was measured in the presence of excess of unlabeled compounds and in fused membrane vesicles loaded with unlabeled compounds to demonstrate their transport. A large number of nucleotides were tested, but only ADP and ATP are suitable substrates for human AAC1, demonstrating a very narrow specificity. Next we tried to understand the molecular basis of this specificity by carrying out molecular-dynamics simulations with selected nucleotides, which were placed at the entrance of the central cavity. The binding of the phosphate groups of guanine and adenine nucleotides is similar, yet there is a low probability for the base moiety to be bound, likely to be rooted in the greater polarity of guanine compared to adenine. AMP is unlikely to engage fully with all contact points of the substrate binding site, suggesting that it cannot trigger translocation.

The first 3′:5′-cyclic nucleotide–amino acid complex:L-His–cIMP

In the crystal structure of the L-His-cIMP complex, i.e. L-histidinium inosine 3':5'-cyclic phosphate [systematic name: 5-(2-amino-2-carboxyethyl)-1H-imidazol-3-ium 7-hydroxy-2-oxo-6-(6-oxo-6,9-dihydro-1H-purin-9-yl)-4a,6,7,7a-tetrahydro-4H-1,3,5,2λ(5)-furo[3,2-d][1,3,2λ(5)]dioxaphosphinin-2-olate], C(6)H(10)N(3)O(2)(+)·C(10)H(10)N(4)O(7)P(-), the Hoogsteen edge of the hypoxanthine (Hyp) base of cIMP and the Hyp face are engaged in specific amino acid-nucleotide (His···cIMP) recognition, i.e. by abutting edge-to-edge and by π-π stacking, respectively. The Watson-Crick edge of Hyp and the cIMP phosphate group play a role in nonspecific His···cIMP contacts. The interactions between the cIMP anions (anti/C3'-endo/trans-gauche/chair conformers) are realized mainly between riboses and phosphate groups. The results for this L-His-cIMP complex, compared with those for the previously reported solvated L-His-IMP crystal structure, indicate a different nature of amino acid-nucleotide recognition and interactions upon the 3':5'-cyclization of the nucleotide phosphate group.

Effect of transition metal ions on the fluorescence and Taq-catalyzed polymerase chain reaction of tricyclic cytidine analogs

The cytosine analogs 1,3-diaza-2-oxophenothiazine (tC) and 1,3-diaza-2-oxophenoxazine (tCo) stand out among fluorescent bases due to their unquenched fluorescence emission in double-stranded DNA. Recently, we reported a method for the generation of densely tCo-labeled DNA by polymerase chain reaction (PCR) that relied on the use of the extremely thermostable Deep Vent polymerase. We have now developed a protocol that employs the more commonly used Taq polymerase. Supplementing the PCR with Mn 2+ or Co 2+ ions dramatically increased the amount of tCo triphosphate (dtCoTP) incorporated and, thus, enhanced the brightness of the PCR products. The resulting PCR products could be easily detected in gels based on their intrinsic fluorescence. The Mn 2+ ions modulate the PCR by improving the bypass of template tCo and the overall catalytic efficiency. In contrast to the lower fidelity during tCo bypass, Mn 2+ improved the ability of Taq polymerase to distinguish between dtCoTP and dTTP when copying a template dA. Interestingly, Mn 2+ ions hardly affect the fluorescence emission of tC(o), whereas the coordination of Co 2+ ions with the phosphate groups of DNA and nucleotides statically quenches tC(o) fluorescence with small reciprocal Stern–Vollmer constants of 10–300 μM.

Nucleotide recognition by protonated aminoglycosides

Interactions of protonated forms of kanamycin A with nucleotides and several simple phosphate anions have been studied by potentiometric and NMR titrations. The affinity of kanamycin A to anions is comparable to that observed with other aliphatic polyammonium receptors of similar charge, but it discriminates triphosphate nucleotides with different nucleobases with binding constants following the order GTP≫CTP ≈ ATP. Kanamycin A also binds the respective uncharged nucleosides with the same selectivity. Binding of ATP is exothermic with a negative entropic contribution in contrast to what is expected for simple ion pairing. Other tested aminoglycosides, amikacin and streptomycin, bind ATP less efficiently than kanamycin A. Models of structures of kanamycin A complexes with ATP and GTP obtained by molecular mechanics (OPLS-2005) calculations based on 1H and 31P NMR data confirm the possibility of nucleotide discrimination by simultaneous ion pairing of terminal nucleotide phosphate groups with ammonium sites of rings B and C and hydrogen bonding of the nucleobase at the ring A of the aminoglycoside.

Real-time NMR Study of Guanine Nucleotide Exchange and Activation of RhoA by PDZ-RhoGEF

Small guanosine triphosphatases (GTPases) become activated when GDP is replaced by GTP at the highly conserved nucleotide binding site. This process is intrinsically very slow in most GTPases but is significantly accelerated by guanine nucleotide exchange factors (GEFs). Nucleotide exchange in small GTPases has been widely studied using spectroscopy with fluorescently tagged nucleotides. However, this method suffers from effects of the bulky fluorescent moiety covalently attached to the nucleotide. Here, we have used a newly developed real-time NMR-based assay to monitor small GTPase RhoA nucleotide exchange by probing the RhoA conformation. We compared RhoA nucleotide exchange from GDP to GTP and GTP analogues in the absence and presence of the catalytic DH-PH domain of PDZ-RhoGEF (DH-PH(PRG)). Using the non-hydrolyzable analogue guanosine-5'-O-(3-thiotriphosphate), which we found to be a reliable mimic of GTP, we obtained an intrinsic nucleotide exchange rate of 5.5 x 10(-4) min(-1). This reaction is markedly accelerated to 1179 x 10(-4) min(-1) in the presence of DH-PH(PRG) at a ratio of 1:8,000 relative to RhoA. Mutagenesis studies confirmed the importance of Arg-868 near a conserved region (CR3) of the Dbl homology (DH) domain and revealed that Glu-741 in CR1 is critical for full activity of DH-PH(PRG), together suggesting that the catalytic mechanism of PDZ-RhoGEF is similar to Tiam1. Mutation of the single RhoA (E97A) residue that contacts the pleckstrin homology (PH) domain rendered the mutant 10-fold less sensitive to the activity of DH-PH(PRG). Interestingly, this mutation does not affect RhoA activation by leukemia-associated RhoGEF (LARG), indicating that the PH domains of these two homologous GEFs may play different roles.

Coordination Reactions and Noncovalent Interactions of Polyamines with Nucleotides in Binary Systems and with Nucleotides and Copper(II) Ion in Ternary Systems.

Interactions of nucleotides (AMP, CMP) and 1,2-diaminopropane (tn-1) or 2-methyl-1,2-diaminopropane (tn-2) in metal-free systems as well as in the systems including copper(II) ions were studied. The composition and overall stability constants of the complexes formed were determined by the potentiometric method, whereas the interaction centres and coordination sites were identified by spectroscopic methods. It was found that phosphate groups of nucleotides and the protonated amine groups of polyamines are the centres of interaction. The differences in the interactions with the polyamines which act as models of biogenic amines are impacted by the presence of lateral chains (methylene groups) in tn-1 and tn-2. In the ternary systems with Cu(II) ions, the heteroligand complexes are mainly of the ML⋯L' type, in which the protonated polyamine is engaged in noncovalent interactions with the anchoring Cu(II)-nucleotide complex. The complexes formed in the Cu/NMP)/tn-1 system are more stable than those formed in the system with tn-2. The mode of coordination in the complex is realised mainly through the phosphate groups of the nucleotide with involvement of the endocyclic nitrogen atoms in a manner which depends upon the steric conditions and in particular on the number of the methylene groups in the polyamine molecule.

Structural basis for VO2+ inhibition of nitrogenase activity (A): 31P and 23Na interactions with the metal at the nucleotide binding site of the nitrogenase Fe protein identified by ENDOR spectroscopy

We previously reported the vanadyl hyperfine couplings of VO(2+)-ATP and VO(2+)-ADP complexes in the presence of the nitrogenase Fe protein from Klebsiella pneumoniae (Petersen et al. in Biochemistry 41:13253-13263, 2002). It was demonstrated that different VO(2+)-nucleotide coordination environments coexist and are distinguishable by electron paramagnetic resonance (EPR) spectroscopy. Here orientation-selective continuous-wave electron-nuclear double resonance (ENDOR) spectra have been investigated especially in the low-radio-frequency range in order to identify superhyperfine interactions with nuclei other than protons. Some of these resonances have been attributed to the presence of a strong interaction with a 31P nucleus although no resolvable superhyperfine structure due to 31P or other nuclei was detected in the EPR spectra. The superhyperfine coupling component is determined to be about 25 MHz. Such a 31P coupling is consistent with an interaction of the metal with phosphorus from a directly, equatorially coordinated nucleotide phosphate group(s). Additionally, novel more prominent 31P ENDOR signals are detected in the low-frequency region. Some of these correspond to a relatively weak 31P coupling. This coupling is present with ATP for all pH forms but is absent with ADP. The ENDOR resonances of these weakly coupled 31P are likely to originate from an interaction of the metal with a nucleotide phosphate group of the nucleoside triphosphate and are attributed to a phosphorus with axial characteristics. Another set of resonances, split about the nuclear Zeeman frequency of 23Na, was detected, suggesting that a monovalent Na+ ion is closely associated with the divalent metal-nucleotide binding site. Na+ replacement by K+ unambiguously confirmed that ENDORs at radio frequencies between 3.0 and 4.5 MHz arise from an interaction with Na+ ions. In contrast to the low-frequency 31P signal, these resonances are present in spectra with both ADP and ATP, and for both low- and neutral-pH forms, although slight differences are detected, showing that these are sensitive to the nucleotide and pH.

Structural Insight into AMPK Regulation: ADP Comes into Play

The AMP-activated protein kinase (AMPK), a sensor of cellular energy status found in all eukaryotes, responds to changes in intracellular adenosine nucleotide levels resulting from metabolic stresses. Here we describe crystal structures of a heterotrimeric regulatory core fragment from Schizosaccharomyces pombe AMPK in complex with ADP, ADP/AMP, ADP/ATP, and 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranotide (AICAR phosphate, or ZMP), a well-characterized AMPK activator. Prior crystallographic studies had revealed a single site in the gamma subunit that binds either ATP or AMP within Bateman domain B. Here we show that ZMP binds at this site, mimicking the binding of AMP. An analogous site in Bateman domain A selectively accommodates ADP, which binds in a distinct manner that also involves direct ligation to elements from the beta subunit. These observations suggest a possible role for ADP in regulating AMPK response to changes in cellular energy status.