Nucleotide Complexes Research Articles

Structural basis for UTP specificity of RNA editing TUTases from Trypanosoma brucei.

Trypanosomatids are pathogenic protozoa that undergo a unique form of post-transcriptional RNA editing that inserts or deletes uridine nucleotides in many mitochondrial pre-mRNAs. Editing is catalyzed by a large multiprotein complex, the editosome. A key editosome enzyme, RNA editing terminal uridylyl transferase 2 (TUTase 2; RET2) catalyzes the uridylate addition reaction. Here, we report the 1.8 A crystal structure of the Trypanosoma brucei RET2 apoenzyme and its complexes with uridine nucleotides. This structure reveals that the specificity of the TUTase for UTP is determined by a crucial water molecule that is exquisitely positioned by the conserved carboxylates D421 and E424 to sense a hydrogen atom on the N3 position of the uridine base. The three-domain structure also unveils a unique domain arrangement not seen before in the nucleotidyltansferase superfamily, with a large domain insertion between the catalytic aspartates. This insertion is present in all trypanosomatid TUTases. We also show that TbRET2 is essential for survival of the bloodstream form of the parasite and therefore is a potential target for drug therapy.

Mn2+-adenosine nucleotide complexes in the presence of the nitrogenase iron-protein: detection of conformational rearrangements directly at the nucleotide binding site by EPR and 2D-ESEEM (two-dimensional electron spin-echo envelope modulation spectroscopy).

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.

Enzyme−Substrate Interactions with an Antibiotic Resistance Enzyme: Aminoglycoside Nucleotidyltransferase(2‘ ‘)-Ia Characterized by Kinetic and Thermodynamic Methods

Aminoglycoside nucleotidyltransferase(2'')-Ia is one of the most often detected enzymes in aminoglycoside-resistant bacteria. Despite its prevalence, little biochemical and biophysical work has been reported for this enzyme. In the current study, substrate specificity and temperature dependence of k(cat) are determined by kinetic assays. Dissociation constants and thermodynamic properties of enzyme-substrate complexes are determined by isothermal titration calorimetry, electron paramagnetic resonance, and fluorescence spectroscopy. Kinetic studies show that aminoglycosides with 2'-NH(2) are better substrates (higher k(cat)/K(m)) than ones with 2'-OH when magnesium(II) is used as the catalytically required divalent cation. The activity is reduced 10-fold for substrates with 2'-NH(2) when manganese(II) replaces magnesium as the required metal. However, kanamycin A, which has a 2'-OH, shows a much smaller decrease in activity when manganese substitutes for magnesium as the divalent cation. Temperature dependence studies show the activation energy of catalysis to be 19.2 kcal/mol and the temperature optimum between 30 and 32 degrees C. The binding of the aminoglycoside substrate tobramycin to the enzyme occurs with a favorable enthalpy which compensates for a large entropic penalty to yield a negative DeltaG value for the complex formation. Enthalpy of binding is less exothermic in the presence of metal-nucleotide. However, due to the more favorable entropy, a more favorable DeltaG is observed for the formation of the enzyme-metal-nucleotide:aminoglycoside complex. Tobramycin binds to ANT(2' ') with a dissociation constant of 0.6 microM, which is further reduced by 3-fold when metal-nucleotide is present. Binding of ATP to the enzyme is determined to be very weak in the absence of a divalent cation, and becomes 2 orders of magnitude tighter when magnesium or manganese is present. Binding studies also show that, in addition to binding to the enzyme in the form of metal-nucleotide complex, a second catalytically required metal binds to an additional site on the enzyme.

A macroscopic kinetic model for DNA polymerase elongation and high-fidelity nucleotide selection

The enzymatically catalyzed template-directed extension of ssDNA/primer complex is an important reaction of extraordinary complexity. The DNA polymerase does not merely facilitate the insertion of dNMP, but it also performs rapid screening of substrates to ensure a high degree of fidelity. Several kinetic studies have determined rate constants and equilibrium constants for the elementary steps that make up the overall pathway. The information is used to develop a macroscopic kinetic model, using an approach described by Ninio [Ninio J., 1987. Alternative to the steady-state method: derivation of reaction rates from first-passage times and pathway probabilities. Proc. Natl. Acad. Sci. U.S.A. 84, 663–667]. The principle idea of the Ninio approach is to track a single template/primer complex over time and to identify the expected behavior. The average time to insert a single nucleotide is a weighted sum of several terms, including the actual time to insert a nucleotide plus delays due to polymerase detachment from either the ternary (template-primer-polymerase) or quaternary (+nucleotide) complexes and time delays associated with the identification and ultimate rejection of an incorrect nucleotide from the binding site. The passage times of all events and their probability of occurrence are expressed in terms of the rate constants of the elementary steps of the reaction pathway. The model accounts for variations in the average insertion time with different nucleotides as well as the influence of G + C content of the sequence in the vicinity of the insertion site. Furthermore the model provides estimates of error frequencies. If nucleotide extension is recognized as a competition between successful insertions and time delaying events, it can be described as a binomial process with a probability distribution. The distribution gives the probability to extend a primer/template complex with a certain number of base pairs and in general it maps annealed complexes into extension products.

Studies of Glutamate Dehydrogenase

1 Equilibrium dialyses have shown tighter binding of GTP to glutamate dehydrogenase in triethanolamine than in phosphate buffer, as characterized by dissociation constants of 35 μM and 325 μM, respectively. If competition between GTP and phosphate occurs, a dissociation constant of 13 mM can be calculated for the binding of phosphate. Binding of ADP, however, is not changed when phosphate buffer is replaced by triethanolamine buffer. 2 The binding of GTP and ADP to the enzyme as well as their mutual interaction in the bound state has also been examined by equilibrium dialyses. Both nucleotides bind simultaneously but with increased dissociation constants. For this negative interaction of ADP and GTP, a coupling energy of ΔG0c=+ 3.3 kJ mol−1 (polypeptide chain)−1 can be calculated. The total number of sites occupied by the two effectors in the ternary complex is identical to the sum of binding sites for the two nucleotides in their respective binary complexes. Thus separate although interacting sites exist for ADP and GTP. 3 Nucleotide binding within the ternary glutamate dehydrogenase-NADPH-GTP complex has been correlated with ligand-induced dissociation of the enzyme to the unimer (the functional subunit comprising six polypeptide chains). Equilibrium dialyses revealed curved Scatchard plots indicating positive cooperative formation of the ternary complex from both binary enzyme-nucleotide complexes. In addition to indicating decreased dissociation constants of the two nucleotides, the binding curves reflect the ligand-induced dissociation of the enzyme into the unimer. Sedimentation analyses of the latter process show close agreement between the degree of ternary complex formation and the extent of dissociation of the enzyme. Two defined states of the enzyme can be distinguished. One state exists as the binary enzyme · nucleotide complex with an unchanged association equilibrium; the other state is completely dissociated into unimers which are saturated with both nucleotides, GTP and NADPH. The exchange between these two states is low. 4 The influence of ADP on the ternary enzyme-NADPH-GTP complex has been studied with respect to both the nucleotide binding and to the association equilibrium of the enzyme. ADP strongly decreases the affinity of the enzyme for both NADPH and GTP and, in addition, restores the ability of the enzyme to associate. The latter process appears to be complete after two or three of the total of six ADP-binding sites per unimer are saturated. The quaternary complex, enzyme-NADPH-GTP-ADP, stabilizes the associated state of the enzyme. However, at high concentration of GTP and ADP, the dissociation constant for NADPH binding to the enzyme is the same as in the enzyme · NADPH binary complex. Thus the effects of two purine nucleotides cancel each other.

Structure of the Nucleotide Complex of PyrR, the pyr Attenuation Protein from Bacillus caldolyticus , Suggests Dual Regulation by Pyrimidine and Purine Nucleotides

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.

Mechanistic Insights into the Suppression of Drug Resistance by Human Immunodeficiency Virus Type 1 Reverse Transcriptase Using α-Boranophosphate Nucleoside Analogs

A class of amino acid substitutions in drug-resistant HIV-1 reverse transcriptase (RT) is responsible for the selectively impaired incorporation of the nucleotide analog inhibitor into DNA. We have shown previously that alpha-boranophosphate nucleoside analogs suppress RT-mediated resistance when the catalytic rate is responsible for drug resistance such as in the case of K65R and dideoxy (dd)NTPs, and Q151M toward AZTTP and ddNTPs. Here, we extend this property to BH3-d4TTP and BH3-3TCTP toward their clinically relevant mutants Q151M and M184V, respectively. Pre-steady-state kinetics on mutants of the Q151M RT family reveal a 3-5-fold resistance to d4TTP. This resistance is suppressed using BH3-d4TTP. Likewise, resistance to 3TCTP by M184V RT (30-fold) and K65R/M184V RT (180-fold) is suppressed using BH3-3TCTP because of a 160-fold acceleration of the catalytic constant kpol. Mechanistic insights into the rate enhancement were obtained using various alpha-boranophosphate nucleotides. The presence of the BH3 group renders kpol independent of amino acid substitutions present in RT. Indeed, the approximately 100-fold decrease in polymerase activity caused by the R72A substitution is restored to wild-type levels using BH3-dTTP. Metal ion titration studies show that alpha-boranophosphate nucleoside analogs enhance 3-8-fold the binding of Mg2+ ions to the active site of the RT.DNA.dNTP complex and alleviate the requirement of critical amino acids involved in phosphodiester bond formation. To our knowledge, this is the first example of rescue of polymerase activity by means of a nucleotide analog.

A novel bis-phenanthridine triamine with pH controlled binding to nucleotides and nucleic acids

The new bis-phenanthridine triamine is characterised by three pK(a) values: 3.65; 6.0 and >7.5. A significant difference in the protonation state of at pH = 5 (four positive charges) and at pH = 7 (less than two positive charges) accounts for the strong dependence of -nucleotide binding constants on nucleotide charge under acidic conditions, whereas at neutral pH all -nucleotide complexes are of comparable stability. All experimental data point at intercalation as the dominant binding mode of to polynucleotides. However, there is no indication of bis-intercalation of the two phenanthridine subunits in binding to double stranded polynucleotides, the respective complexes being most likely mono-intercalative. Thermal stabilisation of calf thymus DNA (ct-DNA) and poly A-poly U duplexes upon addition of is significantly higher at pH = 5 than at neutral conditions. This is not the case with poly dA-poly dT, indicating that the specific secondary structure of the latter, most likely the shape of the minor groove, plays a key role in complex stability. At pH = 5 acts as a fluorimetric probe for poly G (emission quenching) as opposed to other ss-polynucleotides (emission increase), while at neutral conditions this specificity is lost. One order of magnitude higher cytotoxicity of compared to its "monomer" can be accounted for by cooperative action of two phenanthridinium units and the charged triamine linker. The results presented here are of interest to the development of e.g. sequence-selective cytostatic drugs, and in particular for the possibility to control the drug activity properties over binding to DNA and/or RNA by variation of the pH of its surrounding.

Infrared Cavity Ringdown Spectroscopy of Jet-Cooled Nucleotide Base Clusters and Water Complexes

We present the first direct infrared absorption measurements of gas phase nucleotide base clusters and complexes with water. Spectra in the NH stretching region indicate the presence of several doubly H-bonded isomers of both thymine and uracil dimers, as well as both larger base clusters and uracil−water complexes, but no singly H-bonded species are observed. Three NH stretching bands are assigned to three different uracil−water dimer isomers. Bands due to larger complexes are also observed, but due to spectral congestion, assignment to specific clusters is tentative. The IR cavity ringdown laser absorption spectroscopy results show unusually large bonded OH stretch red shifts for uracil−water clusters, providing evidence of the extremely strong hydrogen bonds formed between these molecules predicted in ab initio calculations.

Native UCP1 Displays Simple Competitive Kinetics between the Regulators Purine Nucleotides and Fatty Acids

Elucidation of the regulation of uncoupling protein 1 (UCP1) activity in its native environment, i.e. the inner membrane of brown-fat mitochondria, has been hampered by the presence of UCP1-independent, quantitatively unresolved effects of investigated regulators on the brown-fat mitochondria themselves. Here we have utilized the availability of UCP1-ablated mice to dissect UCP1-dependent and UCP1-independent effects of regulators. Using a complex-I-linked substrate (pyruvate), we found that UCP1 can mediate a 4-fold increase in thermogenesis when stimulated with the classical positive regulator fatty acids (oleate). After demonstrating that the fatty acids act in their free form, we found that UCP1 increased fatty acid sensitivity approximately 30-fold (as compared with the 1.5-fold increase reported earlier based on nominal fatty acid values). By identifying the UCP1-mediated fraction of the response, we could conclude that the interaction between purine nucleotides (GDP) and fatty acids (oleate) unexpectedly displayed simple competitive kinetics. In GDP-inhibited mitochondria, oleate apparently acted as an activator. However, only a model in which UCP1 is inherently active (i.e."activating" fatty acids cannot be included in the model), where GDP functions as an inhibitor with a K(m) of 0.05 mm, and where oleate functions as a competitive antagonist for the GDP effect (with a K(i) of 5 nm) can fit all of the experimental data. We conclude that, when examined in its native environment, UCP1 functions as a proton (equivalent) carrier in the absence of exogenous or endogenous fatty acids.

Purification of RanGDP, RanGTP, and RanGMPPNP by ion exchange chromatography

Ran is a small GTPase that cycles between a guanosine diphosphate (GDP)-bound form (RanGDP) and a guanosine triphosphate (GTP)-bound form (RanGTP) and plays important roles in nuclear transport and mitosis. For studies of Ran function and its interactions with partner proteins, pure RanGDP and RanGTP complexes are critical. Ran complexed with the nonhydrolyzable GTP analog, GMPPNP (RanGMPPNP), is used instead of RanGTP when inhibition of hydrolysis is required. In this study, we demonstrate that the binding of Ran to a UNO Q ion exchange column is remarkably sensitive to small shifts in MgCl 2 concentration, and we use this property to purify recombinant RanGTP, RanGMPPNP, and RanGDP complexes. At 10 mM MgCl 2, Ran was found predominantly in the flow-through and, thus, was separated from the vast majority of bacterial proteins. After reducing the concentration of MgCl 2 to 5 mM, further purification of RanGTP, RanGMPPNP, and RanGDP was achieved by loading onto ion exchange columns and elution with an NaCl gradient. Purity of the resulting preparations was confirmed by releasing the bound nucleotide and checking it against a known nucleotide by high-performance liquid chromatography (HPLC). To further confirm the purity and function of the Ran preparations, appropriate protein-binding, enzymatic, and nuclear import assays were carried out. These methods should facilitate studies of cellular processes involving Ran by providing pure functional Ran–nucleotide complexes.

PH-dependent perturbation of Ras-guanine nucleotide interactions and Ras guanine nucleotide exchange.

p21Ras (Ras) proteins cycle between active GTP-bound and inactive GDP-bound states to mediate signal transduction pathways that promote cell growth, differentiation, and apoptosis. To better understand how cellular regulatory factors, such as guanine nucleotide exchange factors (GEFs) and nitric oxide (NO), modulate Ras-guanine nucleotide binding interactions, we have conducted NMR and kinetic studies to investigate the pH dependence of Ras-GDP interactions and Ras-guanine nucleotide exchange (GNE). pH-sensitive amide protons were identified and found to be associated with residues in the switch I (Phe28-Asp30) and switch II (Asp57 and Thr58) regions of Ras. Furthermore, most of the residues that interact with Mg2+ exhibit pH-sensitive amide proton chemical shifts which appear to be coupled to pH-dependent Ras Mg2+ binding and guanine nucleotide binding affinity. These results suggest that perturbation of Mg2+ interactions within the Ras-guanine nucleotide complex is critical for pH-dependent dissociation of guanine nucleotide ligands from Ras. Notably, these same regions undergo conformational changes upon association with the Ras GEF, SOS. In addition, although we have recently shown that addition of NO to Ras in the presence of oxygen produces a Ras thiyl radical intermediate that promotes Ras GNE, we have also postulated that another byproduct of this reaction, a H+, may contribute to NO-mediated GNE. However, the results presented herein suggest that the H+ byproduct of the reaction is unlikely to be involved in the NO-mediated Ras GNE.

Borate-nucleotide complex formation depends on charge and phosphorylation state.

Flow injection analysis with electrospray ionization mass spectrometry was used to investigate borate-nucleotide complex formation. Solutions containing 100 microM nucleotide and 500 microM boric acid in water-acetonitrile-triethylamine (50:50:0.2, v/v/v; pH 10.3) showed that borate complexation with nicotinamide nucleotides was significantly influenced by the charge on the nicotinamide group and the number of phosphate groups on the adenine ribose. Borate binding decreased in the order of NAD(+), NADH, NADP(+) and NADPH. To investigate the relationship between complex formation and phosphorylation, association constants (K(A)) of borate-adenine (AMP, ADP, ATP), -guanine (GMP, GDP, GTP), -cytidine (CMP, CDP, CTP) and -uridine (UMP, UDP, UTP) complexes were compared. The results showed that the number of nucleotide phosphate groups was inversely proportional to the relative abundance of the borate complexes, with the K(A) of borate-nucleotide complex decreasing in the order mono-, di- and tri-phosphates (AMP approximately GMP approximately CMP approximately UMP > ADP approximately GDP approximately CDP approximately UDP > GTP > ATP approximately CTP approximately UTP). At pH 7.4, using ammonium bicarbonate buffer, only borate-NAD(+) complex was observed. This indicates that the borate-NAD(+) complex may be the most physiologically relevant of those studied.

The 6th International Meeting on Single Nucleotide Polymorphism and Complex Genome Analysis

PharmacogenomicsVol. 5, No. 2 Meeting ReportThe 6th International Meeting on Single Nucleotide Polymorphism and Complex Genome AnalysisAndrew G ClarkAndrew G ClarkMolecular Biology & Genetics, Cornell University, Ithaca, NY 14853, USASearch for more papers by this authorPublished Online:4 Nov 2004https://doi.org/10.1517/phgs.5.2.153.27482AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit FiguresReferencesRelatedDetailsCited ByHuman Genome Variation 2006: emerging views on structural variation and large-scale SNP analysisNature Genetics, Vol. 39, No. 2 Vol. 5, No. 2 Follow us on social media for the latest updates Metrics Downloaded 317 times History Published online 4 November 2004 Published in print March 2004 PDF download

Insertion of an N7-methylguanine mRNA Cap between Two Coplanar Aromatic Residues of a Cap-binding Protein Is Fast and Selective for a Positively Charged Cap

The N7-methylated guanosine (m7G) cap structure, which is found at the 5' ends of mature eukaryotic mRNAs, is critical to a myriad of biological processes. The twenty structures of complexes of cap nucleosides and nucleotides and methylated bases with the vaccinia virus VP39, a cap-specific RNA 2'-O-methyltransferase, which we have determined previously, have revealed the atomic basis of cap binding. The precise insertion and tight fitting of the m7Gua moiety of the cap between two parallel aromatic residues that are spaced only 6.8 A apart governs the high specificity of binding. Here we report the investigation of the reaction mechanism of VP39 with three capped ligands (m7G, m7GpppG, and m7GpppGA3) by fluorescence stopped-flow technique. Cap binding is a simple one-step mechanism with very fast association rate constant (approximately 10(7) M-1 s-1). Moreover, the pH dependence on the association rate constant of m7G binding indicates that only the positively charged keto tautomer of the cap is recognized and bound. The association and dissociation rate constants and affinity constants of the three ligands do not vary greatly, demonstrating that binding is achieved almost entirely by the interactions of m7Gua with two aromatic residues in a cation-pi sandwich.

Crystal Structures of an Archaeal Class I CCA-Adding Enzyme and Its Nucleotide Complexes

CCA-adding enzymes catalyze the addition of CCA onto the 3′ terminus of immature tRNAs without using a nucleic acid template and have been divided into two classes based on their amino acid sequences. We have determined the crystal structures of a class I CCA-adding enzyme from Archeoglobus fulgidus (AfCCA) and its complexes with ATP, CTP, or UTP. Although it and the class II bacterial Bacillus stearothermophilus CCA enzyme (BstCCA) have similar dimensions and domain architectures (head, neck, body, and tail), only the polymerase domain is structurally homologous. Moreover, the relative orientation of the head domain with respect to the body and tail domains, which appear likely to bind tRNA, differs significantly between the two enzyme classes. Unlike the class II BstCCA, this enzyme binds nucleotides nonspecifically in the absence of bound tRNA. The shape and electrostatic charge distribution of the AfCCA enzyme suggests a model for tRNA binding that accounts for the phosphates that are protected from chemical modification by tRNA binding to AfCCA. The structures of the AfCCA enzyme and the eukaryotic poly(A) polymerase are very similar, implying a close evolutionary relationship between them.

The influence of methionine-containing peptides on the reaction of carboplatin with 5′-guanosine monophosphate: a comparison with cisplatin

The pH- and time-dependent reaction of the anticancer drug carboplatin, [Pt(cbdca-κ 2 O, O′)(NH 3) 2] (cbdca=cyclobutane-1,1-dicarboxylate), with the tripeptides H-glyglymet-OH (glycylglycyl- l-methionine) and Ac-glyglymet-OH at 313 K was investigated by high-performance liquid chromatography, NMR and mass spectrometry. The relative stability of the initial ring-opened κ S complex [Pt(cbdca-κ O)(Ac-glyglymet-OH-κ S)(NH 3) 2] leads to increased formation of the kinetically favoured κ S:κ S′ bis-adduct [Pt(Ac-glyglymet-OH-κ S) 2(NH 3) 2] 2+ in comparison with cisplatin. As a result a second 1:2 reaction pathway κ S→κ S:κ S′→κ 2 N M, S:κ S′→κ 3 N G2, N M, S:κ S′, where N M and N G2 represent, respectively, metallated methionine and glycine nitrogen atoms, competes with the 1:1 route κ S→κ 2 N M, S→κ 3 N G2, N M, S also observed for cisplatin. Cleavage of N-acetylglycine at the backbone C(O)–N bond to the second gly residue (G2) is observed after 100 h for the respective tridentate complexes [Pt(Ac-glyglyH −1metH −1-OH-κ 3 N G2, N M, S) (Ac-glyglymet-OH-κ S)] and [Pt(Ac-glyglyH −1metH −1-OH-κ 3 N G2, N M, S)(NH 3)] at pH <5.2. The longevity of the initial κ S complex leads to about an eight-fold increase in the rate of formation of the κ N7:κ N7′ bis-adduct [Pt(5′-GMP-κ N7) 2(NH 3) 2] 2− for the reaction of carboplatin with 5′-GMP 2− at pH 7 in the presence of Ac-glyglymet-OH. A mixed-ligand κ S:κ N7 species [Pt(5′-GMP-κ N7)(Ac-glyglymet-OH-κ S)(NH 3) 2] provides the major precursor for this 1:2 nucleotide complex and κ N7 coordination of 5′-GMP 2− is also observed in the κ 2 N M, S:κ N7 complex [Pt(5′-GMP-κ N7)(Ac-glyglymetH −1-OH-κ 2 N M, S)(NH 3) 2] − formed by substitution of the ammine ligand trans to the methionine sulphur. As the intermediate κ S:κ N7 species is formed rapidly within the first 10 h of reaction, these results suggest that the transfer reaction pathway κ S→κ S:κ N7→κ N7:κ N7′ involving κ S platinated peptides could play an important role in accelerating the rate of DNA binding for carboplatin.

Unexpected binding mode for 2'-phosphoadenosine-based nucleotide inhibitors in complex with human angiogenin revealed by heteronuclear NMR spectroscopy.

Human angiogenin (Ang) is a tumor-promoting RNase in the pancreatic RNase superfamily. Efforts to develop nucleotide-based inhibitors of Ang as potential anticancer drugs have been hampered by the lack of direct structural information on Ang-nucleotide complexes. Here, we have used heteronuclear NMR spectroscopy with (15)N- and (15)N/(13)C-labeled Ang to map the interactions of Ang with the phosphate ion, seven adenosine mononucleotides (the 2'-, 3'-, and 5'-monophosphates, the 2',5'- and 3',5'-diphosphates, the 5'-diphosphate, and the 2'-monophospho-5'-diphosphate), and the dinucleotide 2'-deoxyuridine 3'-pyrophosphate (P' --> 5') adenosine-2'-phosphate (dUppA-2'-p). The 2'-phosphate based derivatives, which bind more tightly than the corresponding 3'-phosphate isomers, induced characteristic large resonance perturbations of the backbone amide proton of Leu(115), the backbone (15)N of His(114), and the Gln(12) side-chain NH(2) group in the Ang active site. In contrast, adenosine derivatives with only 3'- or 5'-phosphates produced much less dramatic perturbations of Leu(115) and His(114) resonances, along with modest perturbations of additional residues both within and beyond the active site. Measurements of NOEs together with molecular docking analyses revealed the three-dimensional structures of the complexes of Ang with adenosine 2',5'-diphosphate and dUppA-2'-p; the binding modes of these inhibitors differ substantially from those predicted in earlier studies. Most notably, the 2'-phosphate rather than the 5'-phosphate occupies the P(1) catalytic subsite of Ang, and the side chain of His(114) has undergone a conformational transition that positions it outside P(1) and allows it to form stacking interactions with the adenine ring of the inhibitor. Strikingly, the 2'-deoxyuridine moiety of dUppA-2'-p makes only a few contacts with Ang, and these involve residues outside the B(1) subsite where the pyrimidine ring of substrates normally binds.

Full-strand Minimization of Single and Double Stranded B-DNA Using Monte-Carlo Annealing

A new algorithm is developed which implements the Monte Carlo annealing method and is used to model and minimize linear, single and double stranded B-DNA sequences. The preliminary B-DNA structures are modeled using initial structures obtained from the Brookhaven database. The model contains structural details at the atomic level and is therefore more elaborate and accurate than the pseudo-atomic and elastomechanical models. The minimization concerns the entire chain length and not only local nucleotide complexes. A variety of DNA sequences (coding or non-coding, random or real, homogeneous or heterogeneous) are investigated in the range of 20-40 base pairs. The potential energy function is written in terms of a set of internal coordinates defined to account for the helical parameters such as twist, tilt and rise, which are important parameters for the description of the global shape of any type of DNA or RNA molecule. The force field used is composed of a limited number of bonded and non-bonded interactions such as bond stretch, angle bend and Lennard-Jones interactions with the Dreiding II force field parameter set used for these interactions. From the minimized structures the angles between Phosphate-Oxygen-Carbon " A 1 " and Oxygen-Phosphate-Oxygen " A 2 " and the average helical twist were calculated. For single strands it is shown that the bond angles are A 1 =107 - 1° and A 2 =122 - 1°, while the helical twist is 37.8 - 1°. For double stranded DNA our model predicts the helical twist of h =35.5 - 2° well, in the A strand, while the prediction is less accurate, h =47 - 2° for the complementary strand B. The average values for the angles A 1 and A 2 are 130 - 1° and 150 - 3° for strand A and 102 - 4° and 123 - 5° for strand B. The reason for this discrepancy is attributed to the different conformations initially adopted by the sugars in the A and B strands.

Studies on the interaction of altromycin B and its platinum(II) and palladium(II) metal complexes with calf thymus DNA and nucleotides

The interaction of the anticancer antibiotic altromycin B and its isostructrural Pt(II) and Pd(II) metal complexes with native calf thymus (CT) DNA was studied using UV–thermal denaturation experiments, circular dichroism spectroscopy and temperature controlled spectrophotometric titrations. Altromycin B stabilizes the double helix by raising the T m, mainly by intercalation of its chromophore between the base pairs and interacting electrostatically via its sugar moieties with the edges of the DNA helix. Moreover, altromycin B induces a B→A structural transition of CT DNA. The effect on DNA stability and conformation depends on the metal ion. Pt(II) and Pd(II) complexes induce the B→A structural transition and stabilize the double helix similarly but they present lower final hyperchromicity due to premelting effects which were caused by intra- and interstrand crosslinking. Thus, a synergic effect of the metal ions to altromycin B–CT DNA interaction is observed in both cases. Altromycin B interacts with 5′-GMP, 5′-AMP and 5′-CMP by electrophilic attack of the opened epoxide ring to the N(7)G, N(1)/N(7)A and N(3)C. Thus, covalent binding between these nucleotides and altromycin B takes place and explain the multiple binding mode suggested by the studies of the interaction of altromycin B and its complexes with DNA. The [Pd(II)–altroB] complex dissociates in the presence of the nucleotides, and various species of Pd(II)–nucleotide complexes, especially with 5′-GMP, are formed. The [Pt(II)–altroB] complex dissociates too, but only one or two species of Pt(II)–nucleotide complexes are formed, and in the case of 5′-AMP interaction the formation of a tertiary altroB–Pt(II)–5′AMP complex is proposed. 5′-TMP reacts very weakly in comparison with the other three nucleotides. These interactions were followed by 1H-NMR.