Nucleotide Recognition Research Articles

Two-Dimensional Arrays of Amphiphilic Zn2+-Cyclens for Guided Molecular Recognition at Interfaces

The sterically guided molecular recognition of nucleobases, phosphates, adenosine, and uridine nucleotides on Langmuir monolayers and Langmuir-Blodgett monolayers of amphiphilic mono- or bis(Zn2+-cyclen)s assembled on thiolated surfaces was investigated. The stepwise selective binding of metal ions, uracil, or phosphate by dicetyl cyclen monolayers with variously tuned structures at the air/water interface was corroborated by the measurements of the corresponding LB films deposited onto quartz crystals. Two types of recognition surfaces were fabricated from Zn2+-dicetyl cyclen. The surface covered with a complex preformed in the Langmuir monolayer was capable both of imide and of phosphate binding. The similar complex formed directly in an LB film on thiolated gold was inactive with respect to imide. The surface plasmon resonance measurements evidenced the stepwise assembly of complementary nucleotides on SAM/LB templates through consecutive phosphate-Zn2+-cyclen coordination. Base pairing between nucleotides resulted in a formation of A-U bilayers comprising two complementary monolayers. Finally, we report on SAM/LB patterns designed for divalent molecular recognition of uridine phosphate by amphiphilic bis(Zn2+-cyclen).

Oxaaza cyclophanes in the recognition of nucleotides. The role of oxygen and electron-rich aromatic rings

Dioxapolyaza cyclophanes derived from resorcinol and different polyamine chains have been studied in aqueous solution as abiotic receptors for nucleotides. The presence of the additional ethyleneoxy subunits is reflected in a higher basicity and in a significant increase in the log K values for the interaction with nucleotides relative to that of related polyazacyclophanes.

Biochemical, biophysical, and proteomic approaches to study DNA helicases

Helicases are a family of enzymes that play an essential role in nearly all DNA metabolic processes, catalyzing the transient opening of DNA duplexes. These motor proteins couple the chemical energy of ATP binding and hydrolysis to the separation of the complementary strands of a DNA or RNA duplex substrate. A full understanding of their mechanism of DNA unwinding can be achieved only through careful investigation of the thermodynamic and kinetic parameters that control this ATP-driven process, as well as through analysis of the helicases' tertiary and quaternary structures associated with nucleic acids and/or nucleotide recognition. This review describes the various biochemical, biophysical, and, more recently, proteomic techniques that have been developed to shed light on the still controversial, and in some aspects elusive, helicase-catalyzed mechanism of DNA unwinding.

Molecular recognition of nucleotides by a new bis(guanidinium)tetrakis(β-cyclodextrin) tetrapod

The facile synthesis of a novel bis(guanidinium)tetrakis(β-cyclodextrin) tetrapod, the first member of a new host family with a pre-defined number of guanidinium centres and cyclodextrins, is presented. Its molecular recognition towards nucleotide guest molecules was investigated. The formation of ditopic bimolecular complexes was observed, combining host–guest hydrophobic inclusion into cyclodextrins and electrostatic interactions between guanidinium and phosphate anions.

ONIOM quantum chemistry study of cyclic nucleotide recognition in phosphodiesterase 5

AbstractCyclic nucleotide phosphodiesterases (PDEs) are enzymes that contribute to the regulation of cyclic nucleotides in the cell by catalyzing the hydrolysis reaction of the O3′‐phosphorous bond, yielding the noncyclic nucleotide as the product. The principal substrates are cyclic 3′,5′‐adenosine and ‐guanosine monophosphate (cAMP and cGMP). PDE5, an important target of drug inhibition, is known to be highly selective for hydrolysis of cGMP. We use all‐quantum hybrid calculations to accurately describe the binding interactions between PDE5 and cAMP/cGMP for the first time. The main reasons for cGMP preference in PDE5 are found to be to the fixed orientation of a conserved glutamine residue (Gln 817) together with the fixed orientation of a nonconserved glutamine residue (Gln 775). We report ONIOM(B3LYP/6‐31g(d):PM3MM) binding energies, which reflect favorable guanine alignment with Gln 817 and steric crowding of adenine by Gln 775. © 2007 Wiley Periodicals, Inc. Int J Quantum Chem, 2007

Nucleotide recognition by the cytoplasmic domain of the human chloride transporter ClC-5

The ubiquitous CBS domains, which are found as part of cytoplasmic domains in the ClC family of chloride channels and transporters, have previously been identified as building blocks for regulatory nucleotide-binding sites. Here we report the structures of the cytoplasmic domain of the human transporter ClC-5 in complex with ATP and ADP. The nucleotides bind to a specific site in the protein. As determined by equilibrium dialysis, the affinities for ATP, ADP and AMP are in the high micromolar range. Point mutations that interfere with nucleotide binding change the transport behavior of a ClC-5 mutant expressed in Xenopus laevis oocytes. Our results establish the structural and energetic basis for the interaction of ClC-5 with nucleotides and provide a framework for future investigations.

X-ray Crystallographic and Steady State Fluorescence Characterization of the Protein Dynamics of Yeast Polyadenylate Polymerase

Polyadenylate polymerase (PAP) catalyzes the synthesis of poly(A) tails on the 3′-end of pre-mRNA. PAP is composed of three domains: an N-terminal nucleotide-binding domain (homologous to the palm domain of DNA and RNA polymerases), a middle domain (containing other conserved, catalytically important residues), and a unique C-terminal domain (involved in protein–protein interactions required for 3′-end formation). Previous X-ray crystallographic studies have shown that the domains are arranged in a V-shape such that they form a central cleft with the active site located at the base of the cleft at the interface between the N-terminal and middle domains. In the previous studies, the nucleotides were bound directly to the N-terminal domain and exhibited a conspicuous lack of adenine-specific interactions that would constitute nucleotide recognition. Furthermore, it was postulated that base-specific contacts with residues in the middle domain could occur either as a result of a change in the conformation of the nucleotide or domain movement. To address these issues and to better characterize the structural basis of substrate recognition and catalysis, we report two new crystal structures of yeast PAP. A comparison of these structures reveals that the N-terminal and C-terminal domains of PAP move independently as rigid bodies along two well defined axes of rotation. Modeling of the nucleotide into the most closed state allows us to deduce specific nucleotide interactions involving residues in the middle domain (K215, Y224 and N226) that are proposed to be involved in substrate binding and specificity. To further investigate the nature of PAP domain flexibility, 2-aminopurine labeled molecular probes were employed in steady state fluorescence and acrylamide quenching experiments. The results suggest that the closed domain conformation is stabilized upon recognition of the correct subtrate, MgATP, in an enzyme-substrate ternary complex. The implications of these results on the enzyme mechanism of PAP and the possible role for domain motion in an induced fit mechanism are discussed.

Epac1 and cAMP-dependent Protein Kinase Holoenzyme Have Similar cAMP Affinity, but Their cAMP Domains Have Distinct Structural Features and Cyclic Nucleotide Recognition

The cAMP-dependent protein kinase (PKA I and II) and the cAMP-stimulated GDP exchange factors (Epac1 and -2) are major cAMP effectors. The cAMP affinity of the PKA holoenzyme has not been determined previously. We found that cAMP bound to PKA I with a K(d) value (2.9 microM) similar to that of Epac1. In contrast, the free regulatory subunit of PKA type I (RI) had K(d) values in the low nanomolar range. The cAMP sites of RI therefore appear engineered to respond to physiological cAMP concentrations only when in the holoenzyme form, whereas Epac can respond in its free form. Epac is phylogenetically younger than PKA, and its functional cAMP site has presumably evolved from site B of PKA. A striking feature is the replacement of a conserved Glu in PKA by Gln (Epac1) or Lys (Epac2). We found that such a switch (E326Q) in site B of human RIalpha led to a 280-fold decreased cAMP affinity. A similar single switch early in Epac evolution could therefore have decreased the high cAMP affinity of the free regulatory subunit sufficiently to allow Epac to respond to physiologically relevant cAMP levels. Molecular dynamics simulations and cAMP analog mapping indicated that the E326Q switch led to flipping of Tyr-373, which normally stacks with the adenine ring of cAMP. Combined molecular dynamics simulation, GRID analysis, and cAMP analog mapping of wild-type and mutated BI and Epac1 revealed additional differences, independent of the Glu/Gln switch, between the binding sites, regarding space (roominess), hydrophobicity/polarity, and side chain flexibility. This helped explain the specificity of current cAMP analogs and, more importantly, lays a foundation for the generation of even more discriminative analogs.

A Basal Promoter Element Recognized by Free RNA Polymerase σ Subunit Determines Promoter Recognition by RNA Polymerase Holoenzyme

During transcription initiation by bacterial RNA polymerase, the sigma subunit recognizes the -35 and -10 promoter elements; free sigma, however, does not bind DNA. We selected ssDNA aptamers that strongly and specifically bound free sigma(A) from Thermus aquaticus. A consensus sequence, GTA(C/T)AATGGGA, was required for aptamer binding to sigma(A), with the TA(C/T)AAT segment making interactions similar to those made by the -10 promoter element (consensus sequence TATAAT) in the context of RNA polymerase holoenzyme. When in dsDNA form, the aptamers function as strong promoters for the T. aquaticus RNA polymerase sigma(A) holoenzyme. Recognition of the aptamer-based promoters depends on the downstream GGGA motif from the aptamers' common sequence, which is contacted by sigma(A) region 1.2 and directs transcription initiation even in the absence of the -35 promoter element. Thus, recognition of bacterial promoters is controlled by independent interactions of sigma with multiple basal promoter elements.

Synthesis of phenanthridinium–bis-nucleobase conjugates, interactions with poly U, nucleotides and in vitro antitumour activity of mono- and bis-nucleobase conjugates

Novel bis-nucleobase–phenanthridinium conjugates were synthesised and their aqueous solutions spectroscopically characterised. Bis-adenine conjugate revealed in aqueous solutions significantly more pronounced intramolecular aromatic stacking interactions than bis-uracil analogue. In contrast with previously reported poly A recognition by bis-uracil conjugate, recognition of complementary nucleotides and poly U was not observed due to the strong interference of bulk water with hydrogen bonding between nucleobases. The screening of anticancer activity on six human cell lines revealed that tethering of a nucleobase to phenanthridinium moiety diminished antiproliferative potential of phenanthridinium. However, among mono-nucleobase conjugates adenine derivative was found to be the most selective one (MiaPaCa–2, Hep–2).

Shuffling of Discrete tRNA Ser Regions Reveals Differently Utilized Identity Elements in Yeast and Methanogenic Archaea

Seryl-tRNA synthetases (SerRSs) from methanogenic archaea possess distinct evolutionary origin and show minimal sequence similarity with counterparts from bacteria, eukaryotes and other archaea. Here we show that SerRS from yeast Saccharomyces cerevisiae and archaeon Methanococcus maripaludis (ScSerRS and MmSerRS, respectively) display significantly different ability to serylate heterologous tRNA Ser. Recognition in yeast was shown to be more stringent than in archaeon. While cross-aminoacylation of M. maripaludis tRNA Ser (MmtRNA Ser) by yeast SerRS barely occurs, yeast tRNA Ser (SctRNA Ser) was shown to be a good substrate for heterologous MmSerRS. To investigate the contribution of different tRNA regions for the recognition by yeast and archaeal SerRS, chimeric tRNAs bearing separated domains of SctRNA Ser in MmtRNA Ser framework were produced by in vitro transcription and subjected to kinetic and gel mobility shift analysis with both enzymes. Generally, the recognition in M. maripaludis seems to be relatively relaxed toward tertiary elements of tRNA Ser structure and relies on the direct recognition of identity nucleotides. On the other hand, expression of tRNA Ser identity elements in yeast seems to be more sensitive toward surrounding sequence context. In both systems variable arm of tRNA was recognized as a major identity region with a strong influence on SerRS:tRNA binding. Acceptor domain of SctRNA Ser was also shown to be important for serylation in yeast. We propose that cognate interactions between N-terminal domain of yeast SerRS and variable region of SctRNA Ser place the acceptor stem into the enzyme's active site and lead to increased affinity toward serine and efficient serylation of tRNA. The same effect was not observed in M. maripaludis. Unlike its yeast counterpart, MmSerRS forms only one type of covalent complex with MmtRNA Ser, regardless of the tRNA/SerRS molar ratio. Stoichiometry of the complex, one tRNA per dimeric SerRS, was revealed by mass spectrometry. Our studies indicate that different SerRS:tRNA recognition mode is utilized by these two systems.

Purification and Initial Biochemical Characterization of ATP:Cob(I)alamin Adenosyltransferase (EutT) Enzyme of Salmonella enterica

ATP:cob(I)alamin adenosyltransferase (EutT) of Salmonella enterica was overproduced and enriched to approximately 70% homogeneity, and its basic kinetic parameters were determined. Abundant amounts of EutT protein were produced, but all of it remained insoluble. Soluble active EutT protein (approximately 70% homogeneous) was obtained after treatment with detergent. Under conditions in which cobalamin (Cbl) was saturating, Km(ATP) = 10 microm, kcat = 0.03 s(-1), and Vmax = 54.5 nm min(-1). Similarly, under conditions in which MgATP was saturating, Km(Cbl) = 4.1 microm, kcat = 0.06 s(-1), and Vmax = 105 nm min(-1). Unlike other ATP:co(I)rrinoid adenosyltransferases in the cell (i.e. CobA and PduO), EutT activity was > or =50-fold higher with ATP versus GTP, and EutT retained 80% of its activity with ADP substituted for ATP and was completely inactive with AMP as substrate, indicating that the enzyme requires the beta-phosphate group of the nucleotide substrate. The data suggest that the amino group of adenine might play a role in nucleotide recognition and/or binding. Unlike the housekeeping CobA enzyme, EutT was not inhibited by inorganic tripolyphosphate (PPPi). Results from 31P NMR spectroscopy studies identified PPi and Pi as by-products of the EutT reaction. In the absence of Cbl, EutT cleaved ATP into adenosine and PPPi, suggesting that PPPi is broken down into PPi and Pi. Electron transfer protein partners for EutT were not encoded by the eut operon. EutT-dependent activity was detected in cell-free extracts of cobA strains enriched for EutT when FMN and NADH were used to reduce cob(III)alamin to cob(I)alamin.

Relations Between Structure and Function of the Mitochondrial ADP/ATP Carrier

Import and export of metabolites through mitochondrial membranes are vital processes that are highly controlled and regulated at the level of the inner membrane. Proteins of the mitochondrial carrier family ( MCF ) are embedded in this membrane, and each member of the family achieves the selective transport of a specific metabolite. Among these, the ADP/ATP carrier transports ADP into the mitochondrial matrix and exports ATP toward the cytosol after its synthesis. Because of its natural abundance, the ADP/ATP carrier is the best characterized within MCF, and a high-resolution structure of one conformation is known. The overall structure is basket shaped and formed by six transmembrane helices that are not only tilted with respect to the membrane, but three of them are also kinked at the level of prolines. The functional mechanisms, nucleotide recognition, and conformational changes for the transport, suggested from the structure, are discussed along with the large body of biochemical and functional results.

Determination of the Amino Acids in Yeast Ribosomal Protein YS11 Essential for the Recognition of Nucleotides in 18 S Ribosomal RNA

The nucleotides in domain I of 18 S rRNA that are important for the binding of the essential yeast ribosomal protein YS11 are mainly in a kink-turn motif and the terminal loop of helix 11 (H11). In the atomic structure of the Thermus thermophilus 30 S subunit, 16 amino acids in S17, the homolog of YS11, are within hydrogen bonding distance of nucleotides in 16 S rRNA. The homologous or analogous 16 amino acids in YS11 were replaced with alanine; nine of the substitutions slowed the growth of yeast cells. The most severe effects were caused by mutations R103A, N106A, K133A, T134A, and K151A. The T. thermophilus analogs of Arg103, Asn106, Thr134, and Lys151 contact nucleotides in the kink-turn motif of 16 S rRNA, whereas Lys133 contacts nucleotides in the terminal loop of H11. These contacts are predominantly with backbone phosphate and sugar oxygens in regions that deviate from A-form geometry, suggesting that YS11 recognizes the shape of its rRNA-binding site rather than reading the sequence of nucleotides. The effect of the mutations on the binding of YS11 to a domain I fragment of 18 S rRNA accorded, in general, with their effect on growth. Mutations of seven YS11 amino acids (Ser77, Met80, Arg88, Tyr97, Pro130, Ser132, and Arg136) whose homologs or analogs in S17 are within hydrogen bonding distance of nucleotides in 16 S rRNA did not affect binding. Apparently, proximities alone do not define either the amino acids or the nucleotides that are important for recognition.

Recognition and Activation of Ryanodine Receptors by Purines

Ryanodine receptor (RyR) is a tetrameric, high molecular weight protein that functions as a calcium release channel. It plays a key role in phenomena such as signal transduction, excitation-contraction and excitation-secretion coupling. Hyperthermia maligna, central core disease and myocardial infarction have been related with RyR dysfunction. RyR is present as three isoforms in vertebrates: RyR 1 mainly localized in skeletal muscle, RyR 2 in cardiac muscle, and RyR 3 in nervous system. RyR is regulated by a number of physiological and pharmacological factors. Main physiological modulators: calcium, kinases and phosphatases, redox state and energy charge. Main pharmacological regulators: caffeine, dantrolene, ruthenium red, heavy metals and ryanodine. Purines have to do with both, physiological and pharmacological regulation of the RyR activity. So far, the mechanisms of RyR activation by ATP and caffeine have been described in detail using [3H]-ryanodine binding assays and unitary channel activity recorded in planar lipid bilayers. However, some questions remain to be addressed and are at present aim of active scrutiny: How many sites for purines are present in the RyR? Is the same site recognized by nucleotides and methylxanthines? What differences exist among the interaction between RyR and purine bases, nucleosides and nucleotides? Are the phosphate groups important for the recognition of nucleotides? Is the sugar moiety important for the recognition of nucleosides? The review article will examine the most recent specialized literature about the mechanism of activation of RyR by purines with emphasis on reports with approaches of structure-function and structure-activation.

Novel inhibition of dATP‐dependent non‐template‐directed nucleotide addition

DNA polymerases play a crucial role in the replication of an organism’s genome. They are essential in the synthesis of DNA using continuous template strands. In addition to conventional synthesis, however, some DNA polymerases are capable of nucleotide addition without template direction. This non-template-directed nucleotide addition (NTA), has many important implications including the possibility for mutations as well as DNA repair. Previous findings point to a significant level of inhibition during NTA when all dNTP’s were present in a reaction, compared to reactions where only dATP was present (Faucett and Islas, Biochem. Biophys. Res. Comm. 337:1030-1037 [2005]). NTA in the presence of only dATP shows high levels of product formation (190nM after 5 min.) whereas in the presence of all four dNTP’s, the rate of NTA is much reduced (70nM after 5 min.) suggesting that one or more nucleotides is inhibiting ATP-dependent NTA (approximately 63% inhibition). This study consisted of an in-depth kinetic analysis of NTA mediated by the 3′-5′ exonuclease deficient large (Klenow) fragment of Escherichia coli DNA Polymerase I. In order to test the rate of NTA, a Cy-5 based oligonucleotide assay was used. In vitro experiments were conducted testing each of three dNTP’s against a dATP concentration gradient. Results from these experiments showed that while dCTP did not inhibit, and dTTP showed minimal levels of inhibition, dGTP data exhibited a significant level of competitive inhibition. Results suggest that dGTP can account for almost all of the inhibition seen during non-template addition in the presence of all dNTP’s. These results have profound significance for understanding the molecular basis of nucleotide recognition by DNA polymerases. This work was supported in part by a grant from the National Science Foundation (NCB – 0315762).

A 'pore' man's sequencing solution?

A protein pore–based system for nucleotide recognition may help make genome sequencing cheaper and easier.

Molecular Basis for Nucleotide-binding Specificity: Role of the Exocyclic Amino Group “N2” in Recognition by a Guanylyl-ribonuclease

Proteins interact with nucleotides to perform a multitude of functions within cells. These interactions are highly specific; however, the molecular basis for this specificity is not well understood. To identify factors critical for protein-guanine nucleotide recognition the binding of two closely related ligands, guanosine 3'-monophosphate (3'GMP) and inosine 3'-monophosphate (3'IMP), to Ribonuclease Sa (RNase Sa), a small, guanylyl-endoribonuclease from Streptomyces aureofaciens, was compared using isothermal titration calorimetry, NMR, X-ray crystallography and molecular dynamics simulations. This comparison has allowed for the determination of the contribution of the exocyclic amino group "N2" of the guanine base to nucleotide binding specificity. Calorimetric measurements indicate that RNase Sa has a higher affinity for 3'GMP (K=(1.5+/-0.2)x10(5)) over 3'IMP (K=(3.1+/-0.2)x10(4)) emphasizing the importance of N2 as a key determinant of RNase Sa guanine binding specificity. This result was unexpected as the published structural data for RNase Sa in complex with 3'GMP showed only a potential long-range interaction (>3.3A) between N2 and the side-chain of Glu41 of RNase Sa. The observed difference in affinity is largely due to a reduction in the favorable enthalpy change by 10 kJ/mol for 3'IMP binding as compared to 3'GMP that is accompanied by a significant difference in the heat capacity changes observed for binding the two ligands. To aid interpretation of the calorimetric data, the first crystal structure of a small, guanylyl ribonuclease bound to 3'IMP was determined to 2.0 A resolution. This structure has revealed small yet unexpected changes in the ligand conformation and differences in the conformations of the side-chains contacting the sugar and phosphate moieties as compared to the 3'GMP complex. The structural data suggest the less favorable enthalpy change is due to an overall lengthening of the contacts between RNase Sa and 3'IMP as compared to 3'GMP. The long-range interaction between N2 and Glu41 is critical for positioning of the nucleotide in the binding cleft for optimal contact formation. Thus, combined, the data demonstrate how a long-range interaction can have a significant impact on nucleotide binding affinity and energetics.

Mutational Analysis of the Catalytic Domain of O-Linked N-Acetylglucosaminyl Transferase

O-Linked N-acetylglucosaminyltransferase (OGT) catalyzes the transfer of O-linked GlcNAc to serine/threonine residues of a variety of target proteins, many of which have been implicated in such diseases as diabetes and neurodegeneration. The addition of O-GlcNAc to proteins occurs in response to fluctuations in cellular concentrations of UDP-GlcNAc, which result from nutrients entering the hexosamine biosynthetic pathway. However, the molecular mechanisms involved in sugar nucleotide recognition and transfer to protein are poorly understood. We employed site-directed mutagenesis to target potentially important amino acid residues within the two conserved catalytic domains of OGT (CD I and CD II), followed by an in vitro glycosylation assay to evaluate N-acetylglucosaminyltransferase activity after bacterial expression. Although many of the amino acid substitutions caused inactivation of the enzyme, we identified three amino acid residues (two in CD I and one in CD II) that produced viable enzymes when mutated. Structure-based homology modeling revealed that these permissive mutants may be either in or near the sugar nucleotide-binding site. Our findings suggest a model in which the two conserved regions of the catalytic domain, CD I and CD II, contribute to the formation of a UDP-GlcNAc-binding pocket that catalyzes the transfer of O-GlcNAc to substrate proteins. Identification of viable OGT mutants may facilitate examination of its role in nutrient sensing and signal transduction cascades.

Unusual sugar nucleotide recognition elements of mesophilic vs. thermophilic glycogen synthases

The glycogen synthase found in Pyrococcus furiosus is a hyperthermophilic biocatalyst that transfers the glucose portion of nucleotide-diphosphoglucose onto a growing carbohydrate biopolymer chain at 80 degrees C. In contrast to the mesophilic rabbit muscle glycogen synthase, the biocatalyst from P. furiosus possesses unusually broad nucleotide tolerance. The enzyme accepts all four common glucose-containing nucleotide-diphosphosugars: ADP-glucose, GDP-glucose, dTDP-glucose, and UDP-glucose. Using an electrospray ionization-mass spectroscopy (ESI-MS) assay, we determined the K(M) and Vmax for GDP-glucose to be 3.9 +/- 0.6 mM and 0.243 +/- 0.009 mM/min, and for dTDP-glucose to be 4.0 +/- 0.5 mM and 0.216 +/- 0.008 mM/min. A related nucleotide sugar, UDP-galactose, was not a reactive substrate, but was instead a competitive inhibitor with a Ki of 17 +/- 2 mM. The glycogen synthase from P. furiosus was shown not to have phosphorylase activity. The DeltaDeltaG of substrate binding was compared between the mesophilic rabbit muscle and the hyperthermophilic P. furiosus glycogen synthase to dissect any differences in sugar nucleotide recognition strategies at elevated temperatures. Both biocatalysts were shown to gain most of their substrate affinity through electrostatic interactions between the enzyme and the alpha-phosphate.