A possible recognition mode of mRNA cap terminal structure by peptide: cooperative stacking and hydrogen-bond pairing interactions between m7GpppA and Trp-Leu-Glu

1H-NMR and fluorescence spectroscopic studies on the interaction between the Trp-Leu-Glu and m7GpppA have shown a specific binding mode, in which the pi-pi stacking interaction of the Trp indole ring and the hydrogen-bond pairing of Glu carboxyl side group with 7-methylguanine base are simultaneously formed.     

Cooperative stacking and hydrogen bond pairing interactions of fragment peptide in cap binding protein with mRNA cap structure

The stacking and hydrogen bonding abilities of Trp-(Gly)n-Glu (n = 0 approximately 3) for the interaction with 7-methylguanine (m7G) base were examined by fluorescence and 1H-NMR methods, and it was shown that they correlate with the distance between the Trp and Glu residues, and become most significant when both residues are separated from each other by two Gly residues (n = 2). Based on this insight, the sequence conserved between the human and yeast cap binding proteins (CBPs) was surveyed, and the sequence of Trp-Glu-Asp-Glu (No. 102-105 in human CBP) was selected as a probable site for the binding with mRNA cap structure. Thus, the stacking and hydrogen bonding abilities of Trp-Glu-Asp-Glu with m7G cap structure were examined by comparative experiments using its analogous peptides. The results showed that the fourth Glu residue is important not only for the construction of hydrogen bond pairing with m7G base but also for strengthening the stacking interaction between the Trp indole ring and m7G base. Taking account of the recognition analysis using the mutant CBP proteins by site-directed mutagenesis (Ueda, H., Iyo, H., Doi, M., Inoue, M., Ishida, T., Morioka, H., Tanaka, T., Nishikawa, S. and Uesugi, S. (1991) FEBS Lett. 280, 207-210), this cooperative interaction could be important for the recognition of mRNA cap structure.     

Inter- and intramolecular stacking interaction between indole and adeninium rings

Crystals of 1,9-dimethyladeninium-indole-3-acetate (1:1) complex (I) and 9-(3-indol-3-ylpropyl)-1-methyladeninium iodide (II), an inter- or intramolecular model for the stacking interaction between the tryptophanyl residue and the methylated (or protonated) adenine base, were subjected to X-ray analyses. Nearly parallel stacking and interplanar spacing near to 3.4 A were observed between the indole and adeninium rings of both crystals. In particular, one of the two stacking pairs formed in I showed the existence of a partial charge-transfer interaction in their ground states. On the basis of the molecular orbital consideration, the mutual orientation between these stacked aromatic rings is considerably governed by the orbital interaction between the highest occupied molecular orbital of the indole ring and the lowest unoccupied one of the adeninium ring. The ring stacking observed in II was stabilized by the strong coupled dipole-dipole interaction. Absorption, fluorescence, and proton nuclear magnetic resonance spectra indicated the existence of a stacking interaction in the aqueous solutions of I and II, as well as in their crystalline states. The biological implication for the observed stacking interactions has been discussed.     

Conformational analysis of the tetranucleotides m6(2)A-m6(2)A-U-m6(2)A(m6(2)A = N6-dimethyladenosine) and U-m6(2)A-U-m6(2)A and of the hybrid dA-r(U-A). A one- and two-dimensional NMR study

A 1H-NMR investigation was carried out on the tetranucleotides U-m6(2)A-U-m6(2)A and m6(2)A-m6(2)A-U-m6(2)A (m6(2) = N6-dimethyladenosine) as well as on the hybrid trinucleotide dA-r(U-A). An extensive comparison with m6(2)A-U-m6(2)A and other relevant compounds is made. Previous proton NMR studies on trinucleotides have shown that purine-pyrimidine-purine sequences prefer to adopt a mixture of states which have as a common feature that the interior pyrimidine residue bulges out, whereas the flanking purine residues stack upon each other. A stacking interaction on the 3' side of the bulge is known to have no measurable effect on the bulge population. Chemical-shift data, ribose ring conformational analysis and information from NOE experiments now show unambiguously that the moderate U(1)-m6(2)A(2) stack in U-m6(2)A-U-m6(2)A diminishes the population of bulged-out structures in favour of a regular stack. This tendency towards conformational transmission in the downstream 5'----3' direction is fully confirmed by the fact that the strong m6(2)A(1)-m6(2)A(2) stack in the tetranucleotide m6(2)A-m6(2)A-U-m6(2)A virtually precludes the formation of bulged-out structures. The conformational characteristics of dA-r(U-A) appear comparable with those of m6(2)A-U-m6(2)A, which indicates that the presence of a 2'-hydroxyl group in the first purine residue is not a necessary prerequisite for the formation of a bulge.     

A role of elementary interactions between nucleic-acid base and amino-acid side chains in specificity of ribonuclease

Several X-ray structures of model crystals that contain hydroxyl group and nucleic-acid bases suggest that hydroxyl group interacts preferentially with pyrimidines than with purines through hydrogen bonds. This explains a role of Thr 45 and Ser 123 at the B1 site of RNAase A. The stacking energies of nucleic-acid bases with the protonated imidazolyl group are estimated to be in the order of C greater than A greater than G greater than U from 1H-NMR spectra and CNDO/2 calculations. Such interactions, in addition to hydrogen bondings, would stabilize the binding of substrates at the B2 site.     

Prominent stacking interaction with aromatic amino acid by N-quarternization of nucleic acid base: X-ray crystallographic characteristics and biological implications

In order to investigate the mode of interaction between the N-quarternized cytosine base and the aromatic amino acid, the crystal structure of the 3-methyl-cytidine-5'-monophosphate:tryptamine complex was analyzed by X-ray diffraction. The complex crystals were stabilized by extensive hydrogen bond formations in which eight independent water molecules per complex pair participated. A prominent stacking interaction, characterized by a parallel alignment of both rings with a separation distance of ca. 3.4 A, was observed between the cytosine base and the indole ring. Combining the present results with X-ray crystallographic data on the adenine--and guanine--aromatic amino acid interactions, we summarize the structural characteristics observed in the stacking interaction of the N-quarternized nucleic acid base with the aromatic amino acid and discuss their biological implications, especially in connection with the significance of N-protonation of nucleic acid base for selective recognition by protein.     

A selective recognition mode of a nucleic acid base by an aromatic amino acid: L-phenylalanine-7-methylguanosine 5''-monophosphate stacking interaction.

The conformation of 7-methylguanosine 5'-monophosphate (m7GMP) and its interaction with L-phenylalanine (Phe) have been investigated by X-ray crystallographic, 1H-nuclear magnetic resonance, and energy calculation methods. The N(7) methylation of the guanine base shifts m7GMP toward an anti--gauche, gauche conformation about the glycosyl and exocyclic C(4')-C(5') bonds, respectively. The prominent stacking observed between the benzene ring of Phe and guanine base of m7GMP is primarily due to the N(7) guarternization of the guanine base. The formation of a hydrogen bonding pair between the anionic carboxyl group and the guanine base further stabilizes this stacking interaction. The present results imply the importance of aromatic amino acids as a hallmark for the selective recognition of a nucleic acid base.     

Computational evaluation of intermolecular interactions of a universal base 3-nitropyrrole in stacked dimers and DNA duplexes

The stacking interactions between a universal base of 3-nitropyrrole (3NP) and four canonical nucleobases were studied by means of ab initio molecular orbital calculations. The stabilities of the complexes are comparable to those of the stacked dimers of canonical bases reported previously. The detailed analysis of the interaction energies revealed the importance of the dipole-dipole interaction included in the Hartree-Fock terms to determine the geometry dependence of the stacking energies. It was also clarified that the dispersion energies included in the electron-correlation terms were essential to obtain adequate stabilities. The contribution of the nitro group was evaluated by the comparative studies of pyrrole and 3NP. The increased molecular dipole moment and surface are expected to account for the enhancement of the stability of the stacked dimers containing 3NP. The force field parameters required for calculation of the molecular mechanics of 3NP were obtained for 3NP on the basis of these molecular orbital calculations. The energy-minimized structures obtained by the molecular mechanics calculations of 3NP accorded with those obtained by the molecular orbital calculations described above. A DNA duplex structure containing 3NP-A, 3NP-T, or 3NP-C was calculated by use of these force field parameters. In the case of 3NP-A, the computationally calculated structure was in good agreement with that previously determined by use of (1)H-NMR except for the orientation of the nitro group.     

The crystal and molecular structure of 1-methyl-3-carbamoyl-pyridinium:indole-3-acetic acid (1:1) monohydrate complex. A model for the intermolecular stacking interaction between the indole ring and NAD+.

The crystal and molecular structure of the title complex has been determined by X-ray diffraction methods. The crystals contain one water molecule per asymmetric unit, which plays a important role in the molecular packing by forming hydrogen bonds with two carboxyl oxygen atoms of indole-3-acetic acid and a carbamoyl nitrogen atom of the 1-methyl-3-carbamoylpyridinium cation. Prominent stacking between the indole ring and the pyridinium ring, caused by the π_D?π_A interaction, is observed. This overlap with substantial result may provide a model for the stacking interaction between NAD⁺ and the tryptophanyl residues in the proteins.     

Effects of essential carbohydrate/aromatic stacking interaction with Tyr100 and Phe259 on substrate binding of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp. 1011

The stacking interaction between a tyrosine residue and the sugar ring at the catalytic subsite -1 is strictly conserved in the glycoside hydrolase family 13 enzymes. Replacing Tyr100 with leucine in cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. 1011 to prevent stacking significantly decreased all CGTase activities. The adjacent stacking interaction with both Phe183 and Phe259 onto the sugar ring at subsite +2 is essentially conserved among CGTases. F183L/F259L mutant CGTase affects donor substrate binding and/or acceptor binding during transglycosylation [Nakamura et al. (1994) Biochemistry 33, 9929-9936]. To elucidate the precise role of carbohydrate/aromatic stacking interaction at subsites -1 and +2 on the substrate binding of CGTases, we analyzed the X-ray structures of wild-type (2.0 A resolution), and Y100L (2.2 A resolution) and F183L/F259L mutant (1.9 A resolution) CGTases complexed with the inhibitor, acarbose. The refined structures revealed that acarbose molecules bound to the Y100L mutant moved from the active center toward the side chain of Tyr195, and the hydrogen bonding and hydrophobic interaction between acarbose and subsites significantly diminished. The position of pseudo-tetrasaccharide binding in the F183L/F259L mutant was closer to the non-reducing end, and the torsion angles of glycosidic linkages at subsites -1 to +1 on molecule 1 and subsites -2 to -1 on molecule 2 significantly changed compared with that of each molecule of wild-type-acarbose complex to adopt the structural change of subsite +2. These structural and biochemical data suggest that substrate binding in the active site of CGTase is critically affected by the carbohydrate/aromatic stacking interaction with Tyr100 at the catalytic subsite -1 and that this effect is likely a result of cooperation between Tyr100 and Phe259 through stacking interaction with substrate at subsite +2.     

Computational Evaluation of Intermolecular Interactions of a Universal Base 3-Nitropyrrole in Stacked Dimers and DNA Duplexes

Abstract

The stacking interactions between a universal base of 3-nitropyrrole (3NP) and four canonical nucleobases were studied by means of ab initio molecular orbital calculations. The stabilities of the complexes are comparable to those of the stacked dimers of canonical bases reported previously. The detailed analysis of the interaction energies revealed the importance of the dipole-dipole interaction included in the Hartree-Fock terms to determine the geometry dependence of the stacking energies. It was also clarified that the dispersion energies included in the electron-correlation terms were essential to obtain adequate stabilities. The contribution of the nitro group was evaluated by the comparative studies of pyrrole and 3NP. The increased molecular dipole moment and surface are expected to account for the enhancement of the stability of the stacked dimers containing 3NP. The force field parameters required for calculation of the molecular mechanics of 3NP were obtained for 3NP on the basis of these molecular orbital calculations. The energy-minimized structures obtained by the molecular mechanics calculations of 3NP accorded with those obtained by the molecular orbital calculations described above. A DNA duplex structure containing 3NP-A, 3NP-T, or 3NP-C was calculated by use of these force field parameters. In the case of 3NP-A, the computationally calculated structure was in good agreement with that previously determined by use of ¹H-NMR except for the orientation of the nitro group.     

X-ray structure of cytidine-5'-O-dimethylphosphate. Novel stacking between the ribosyl O(2') hydroxyl oxygen atom and the base.

The anionic oxygen atoms of the phosphodiester backbone of RNA and DNA are particularly susceptible to esterification by many mutagenic and carcinogenic alkylating agents. To better understand the geometric, electronic and conformational properties of the alkylated sugar phosphate moiety, the X-ray structure of the phosphotriesterified nucleotide, cytidine-5'-O-dimethylphosphate (C11H18N3O8P), was undertaken. The compound crystallizes in the monoclinic space group P2, with unit cell parameters of a = 5.741(2), b = 11.625(1), c = 11.425(1)A, beta = 94.43(2) degrees. The structure was solved by direct methods and refined by block-diagonal least-squares technique to an R index of 0.034 (Rw = 0.046). The D-ribofuranosyl ring is in the 3T2 twist conformation (P = 13.1(2) degrees, tau m = 36.7(2) degrees) and the conformation about the C(1')-N(1) glycosyl bond is anti (XCN = 8.3(2) degrees). The four P-O bond lengths are significantly shorter than those of the nonalkylated nucleotides. The three sets of phosphodiester linkages, (omega 'A, omega A), (omega 'B, omega B) and (omega 'C, omega C), take the (g-,t), (t,g) and (g-,t) conformations, respectively. There is no base-base or alkyl-base stacking, however, a novel intermolecular stacking is found between the ribosyl O(2') hydroxyl oxygen atom and a neighboring pyrimidine ring. This hydroxyl-base stacking interaction may have implications in the stabilization of the tertiary and quarternary structure of ribonucleic acids and nucleic acid-protein complexes.     

Thermodynamical model for insertion and aggregate binding of caffeine to the homopolymer poly(riboadenylate) and model choice by data analysis

This paper describes the model used to estimate the parameters of caffeine-poly(riboadenylate) (poly(A)) interactions from corresponding 1H-NMR measurements. The model of insertion and aggregate binding describes the non-cooperative insertion of a molecule C into an interspace between two monomers of a homopolymer in competition with aggregate binding. It contains two binding constants, K1 for insertion and K2 for the interaction of monomeric A units of the polymer with C molecules in bound aggregates, and two cooperativity parameters, Kcc for stacking of C molecules within aggregates and tau which is thought to be due to conformational adaptation of the polymer to those bound aggregates which cover more than one A unit. In contrast to other models, the size of a binding site (within the aggregates) is less than one monomeric unit, with n denoting the maximum number of C molecules per A unit in bound aggregates. The model is developed for general n by means of the method of sequence-generating functions. For n = 2 and n = 3, the correctness of the model treatment was checked by the matrix method. The model is applicable to the binding of aggregates to homopolymers, which are flexible enough to fit their structure to the aggregates.     

Influence of uracil on the conformational behaviour of RNA oligonucleotides in solution

NMR studies were carried out on some alternating pyrimidine-purine sequences: the single-stranded tetramers CACA and UGUG and the self-complementary octamer CACAUGUG. Assignments, based upon COSY, homonuclear Hartmann-Hahn, and NOESY experiments, are given for the resonances of all base protons and of several sugar protons. Chemical shift vs temperature profiles were used to obtain thermodynamic parameters for the single-stranded stack in equilibrium with random coil and the duplex in equilibrium with random coil equilibria. The populations of N-type conformer of the ribose rings were estimated from the observed J1'2'. Comparisons with another alternating pyrimidine-purine sequence Um2(6)AUm2(6)A and with the deoxyribose counterparts d(CACA), d(TGTG) and d(CACATGTG) are given. Previous 1H-NMR investigations of Um2(6)AUm2(6)A revealed that the population of bulge-out structure diminishes compared to m2(6)AUm2(6)A due to the U(1)-m2(6)A(2) stacking interaction. In CACA a strong stacking proclivity (Tm = 310 K) together with a clear preference for N-type ribose is observed. However, the stacking interactions in UGUG are relatively less stable (Tm = 288 K) and a bias towards S-type sugar is present. Besides a small amount of stack, a significant contribution of bulge out structure is proposed for UGUG. We conclude that the nature of the pyrimidine base mainly determines the formation of bulge-out structures. The poor stacking properties of uracil now appear to be mainly responsible for this phenomenon. Comparison with the deoxyribose counterparts shows a reasonable agreement between the Tm values of CACA and d(CACA), whereas the Tm of UGUG (288 K) is much lower than the Tm of d(TGTG) (315 K). It is suggested that the absence of bulge-out structures in DNA purine-pyrimidine-purine sequences is related to the relatively strong stacking proclivity of dT residues compared to that of U residues. The Tm values (average 341 K) for the duplex in equilibrium with random coil transition obtained for each residue of CACAUGUG appear very similar. All ribose rings, except the G(8), adopt a pure N conformer in the duplex. This is taken to mean that the differences in conformational behaviour of the constituent tetramers disappear upon duplex formation.     

Crystalline structure of N-(S)-2-heptyl (1R,2R)-2-(2,3-anthracenedicarboximido)cyclohexanecarboxamide that differs from its preferred conformation in the solvent used for crystallization

The crystalline structure of N-(S)-2-heptyl (1R,2R)-2-(2,3-anthracenedicarboximido)cyclohexamide (1), which was crystallized from methanol, was determined by an X-ray analysis and had a different conformation from its preferred one in CD3OD by a 1H-NMR analysis. Inter- and intra-molecular CH-pi interaction in a crystal plays a very important role in crystal packing. The preferred conformation of the amide derivative in a solution allows us to exploit (1R,2R)-2-(2,3-anthracenedicarboximido)cyclohexanecarbonyl chloride as a conversion reagent to determine the absolute configuration of chiral amines by 1H-NMR.     

The chirality breaking in the photolysis of leucine enantiomers and 5′CMP

The inhibition of the photohydrate formation of the cytidine 5 monophosphate (5 CMP) under UV irradiation in the presence of the D-, L-leucine in phosphate aqueous solution (pH 7) has been studied. L-leucine demonstrates more effective in preventing 5 CMP from photohydration than that of D-leucine. This photolysis method can be used instead of NMR spectroscopy to research weak nucleotide-amino acid interaction in aqueous solution especially for nonaromatic acid. A statistical thermodynamic stacking model is proposed to describe the mechanism producing chiral symmetry breaking. The calculated weak interaction energy is 1.16kT and 2.05kT for D-, and L-leucine respectively.     

Locations of local anesthetic dibucaine in model membranes and the interaction between dibucaine and a Na+ channel inactivation gate peptide as studied by 2H- and 1H-NMR spectroscopies.

To study the molecular mechanisms of local anesthesia, locations of local anesthetic dibucaine in model membranes and the interactions of dibucaine with a Na+ channel inactivation gate peptide have been studied by 2H- and 1H-NMR spectroscopies. The 2H-NMR spectra of dibucaine-d9 and dibucaine-d1, which are deuterated at the butoxy group and at the 3 position in its quinoline ring, respectively, have been observed in multilamellar dispersions of the lipid mixture composed of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine. 2H-NMR spectra of deuterated palmitic acids incorporated, as a probe, into the lipid mixture containing cholesterol have also been observed. An order parameter, SCD, for each carbon segment was calculated from the observed quadrupole splittings. Combining these results, we concluded that first, the butoxy group of dibucaine is penetrating between the acyl chains of lipids in the model membranes, and second, the quinoline ring of dibucaine is located at the polar region of lipids but not at the hydrophobic acyl chain moiety. These results mean that dibucaine is situated in a favorable position that permits it to interact with a cluster of hydrophobic amino acids (Ile-Phe-Met) within the intracellular linker between domains III and IV of Na+ channel protein, which functions as an inactivation gate. To confirm whether the dibucaine molecule at the surface region of lipids can really interact with the hydrophobic amino acids, we synthesized a model peptide that includes the hydrophobic amino acids (Ac-GGQDIFMTEEQK-OH, MP-1), the amino acid sequence of which corresponds to the linker part of rat brain type IIA Na+ channel, and the one in which Phe has been substituted by Gln (MP-2), and measured 1H-NMR spectra in both phosphate buffer and phosphatidylserine liposomes. It was found that the quinoline ring of dibucaine can interact with the aromatic ring of Phe by stacking of the rings; moreover, the interaction can be reinforced by the presence of lipids. In conclusion, we wish to propose that local anesthesia originates from the pi-stacking interaction between aromatic rings of an anesthetic molecule located at the polar headgroup region of the so-called boundary lipids and of the Phe in the intracellular linker between domains III and IV of the Na+ channel protein, prolonging the inactivated state and consequently making it impossible to proceed to the resting state.     

Ternary copper(II) complexes with N-carboxymethyl-l-prolinato(2-) ion and imidazole or creatinine: a comparative study of the interligand interactions influencing the molecular recognition and stability

The compounds {[Cu(CMP)(Him)].H(2)O}(n) (I) and [Cu(CMP)(crea)H(2)O].3H(2)O (II) were synthesized and characterized by X-ray diffraction, thermal, spectral and magnetic methods (CMP=N-carboxymethyl-;l-prolinato(2-) ion, Him=imidazole and crea=creatinine). Appropriate structural comparison with other compounds such as {[Cu(CMP)(H(2)O)].H(2)O}(n), [Cu(crea)(2)Cl(2)] and [Cu(dipeptide)(crea)(H(2)O)(x)].nH(2)O (x=0 or 1) have been made in order to prove that crea can act as an imidazole-like ligand (because it is able to promote the same fac- to mer-CMP tridentate conformational change in copper(II) complexes) as well as to discuss the interligand interactions which control the 'Cu(CMP) complex-crea, molecular recognition processes. In contrast to that found in related ternary complexes, we have concluded that direct CMP-crea interligand interactions are missing in the Cu-CMP-crea complex due to the inappropriate correspondence between the donor and/or acceptor H-bonding properties of these ligands. CMP can only act as H-acceptor by its two terminal carboxylate group, and crea can display H-donor and H-acceptor roles by its exocyclic -NH(2) and O moieties, respectively. That promotes the reinforcement of the Cu-N(crea) bond by a bridge -N-H(crea)...O(aqua) (2.867(3)A, 176.4 degrees).     

Differential Effects of Lithium on Muscarinic Cholinoceptor-Stimulated CMP-Phosphatidate Accumulation in Cerebellar Granule Cells, CHO-M3 Cells, and SH-SY5Y Neuroblastoma Cells

Abstract: The ability of lithium to potentiate muscarinic cholinoceptor-stimulated CMP-phosphatidate (CMP.PA) accumulation has been examined in various cells in which muscarinic cholinoceptor agonists evoke a phosphoinositide response. Cell types examined include rat cerebellar granule cells, Chinese hamster ovary cells transfected to express the human muscarinic M3 receptor (CHO-M3 cells), and SH-SY5Y neuroblastoma cells. Neither carbachol (1 m M ) nor lithium (10 m M ) caused significant increases in CMP.PA accumulation in rat cerebellar granule cells; however, when added together for 20 min a linear 17-fold increase over basal levels was observed. The increase was dependent on the concentration of carbachol and lithium present, and the effect could be reversed by addition of exogenous myo -inositol (10 m M ). Addition of carbachol alone to CHO-M3 cells caused a five-fold increase in CMP.PA accumulation. In the presence of lithium, a 70-fold increase was observed at 20 min after carbachol plus lithium addition. This latter response was concentration dependent and could be abolished by preincubation in the presence of 10 m M myo -inositol. In contrast, whereas carbachol elicited a three-fold increase in CMP.PA accumulation in SH-SY5Y neuroblastoma cells, which reached a plateau 10 min after agonist addition, the response could neither be augmented by addition of lithium nor inhibited by addition of myo -inositol. These results emphasise that the ability of lithium to affect agonist-stimulated CMP.PA accumulation is not simply a function of stimulus strength, but is also crucially dependent on the intracellular concentration of inositol.     

An X-ray evidence for the ring-stacking interaction between indole ring and the pyrimidine moiety of thiamin: Crystal structure of thiamin indole-3-propionate

The crystal structure of thiamin indole-3-propionate was determined by X-ray diffraction as a model for the possible thiamin coenzyme-tryptophan residue interaction at the binding site of thiamin pyrophosphate dependent enzymes. There is an intermolecular stacking interaction of the indole ring with the pyrimidine ring, but not with the positively charged thiazolium ring, of thiamin retaining the characteristic F-conformation. Although this association is due to dipole-dipole interaction between both aromatic rings, charge-transfer interaction cannot be ruled out in solution state because the absorption spectrum shows the characteristic charge-transfer band.