Fibre X-Ray and Conformational Study of the Binding of Metal Ions on DNA

Abstract

Results obtained from X-ray diffraction as well as from conformational analysis of Ag-DNA fibres are presented. For small percentages of Ag⁺ bound and high humidity, the B-DNA form is maintained. As the percentage of Ag⁺ is increased, the helical parameters of the B-DNA are modified. These modifications are directly related to the percentage of G—C bases. The periodicity of the DNA fibres are perturbed as Ag⁺ is mainly bound to G—C pairs and, thus, only the equatorial diffracted intensities can be compared to values calculated from molecular models. It is shown, by this way, that the first binding site is located on N7 of G. A second site is situated between N3 and N1 of the G—C pair, at the place of a hydrogen bond. A molecular model of the Ag-DNA complex is proposed and shown to be in agreement with experimental data. Results obtained allow to get some information on the binding of other ions such as Cu²⁺ and Hg²⁺ which give very little modification of the fibre X-ray patterns.     

Silver(I) complexes with DNA and RNA studied by Fourier transform infrared spectroscopy and capillary electrophoresis

Ag(I) is a strong nucleic acids binder and forms several complexes with DNA such as types I, II, and III. However, the details of the binding mode of silver(I) in the Ag-polynucleotides remains unknown. Therefore, it was of interest to examine the binding of Ag(I) with calf-thymus DNA and bakers yeast RNA in aqueous solutions at pH 7.1-6.6 with constant concentration of DNA or RNA and various concentrations of Ag(I). Fourier transform infrared spectroscopy and capillary electrophoresis were used to analyze the Ag(I) binding mode, the binding constant, and the polynucleotides' structural changes in the Ag-DNA and Ag-RNA complexes. The spectroscopic results showed that in the type I complex formed with DNA, Ag(I) binds to guanine N7 at low cation concentration (r = 1/80) and adenine N7 site at higher concentrations (r = 1/20 to 1/10), but not to the backbone phosphate group. At r = 1/2, type II complexes formed with DNA in which Ag(I) binds to the G-C and A-T base pairs. On the other hand, Ag(I) binds to the guanine N7 atom but not to the adenine and the backbone phosphate group in the Ag-RNA complexes. Although a minor alteration of the sugar-phosphate geometry was observed, DNA remained in the B-family structure, whereas RNA retained its A conformation. Scatchard analysis following capillary electrophoresis showed two binding sites for the Ag-DNA complexes with K(1) = 8.3 x 10(4) M(-1) for the guanine and K(2) = 1.5 x 10(4) M(-1) for the adenine bases. On the other hand, Ag-RNA adducts showed one binding site with K = 1.5 x 10(5) M(-1) for the guanine bases.     

The molecular structure of the complex of Hoechst 33258 and the DNA dodecamer d(CGCGAATTCGCG).

The crystal structure of the complex between the dodecamer d(CGCGAATTCGCG) and a synthetic dye molecule Hoechst 33258 was solved by X-ray diffraction analysis and refined to an R-factor of 15.7% at 2.25 A resolution. The crescent-shaped Hoechst compound is found to bind to the central four AATT base pairs in the narrow minor groove of the B-DNA double helix. The piperazine ring of the drug has its flat face almost parallel to the aromatic bisbenzimidazole ring and lies sideways in the minor groove. No evidence of disordered structure of the drug is seen in the complex. The binding of Hoechst to DNA is stabilized by a combination of hydrogen bonding, van der Waals interaction and electrostatic interactions. The binding preference for AT base pairs by the drug is the result of the close contact between the Hoechst molecule and the C2 hydrogen atoms of adenine. The nature of these contacts precludes the binding of the drug to G-C base pairs due to the presence of N2 amino groups of guanines. The present crystal structural information agrees well with the data obtained from chemical footprinting experiments.     

Circular dichroism calculations for double-stranded polynucleotides of repeating sequence

Optical property calculations are presented for poly(A·U), poly[(A-U)·(A-U)], poly(G·C), and poly[(G-C)·(G-C)] in RNA, B-DNA, and C-DNA conformations. An all-order classical coupled oscillator polarizability theory was used, and an effective dielectric constant of 2 was assumed. The calculated CD spectra were found to be sensitive to both geometry and sequence. Agreement with the measured CD spectra of poly(A·U), poly(G·C), and poly(dG·dC) is very good. Calculations for other sequences and geometries are less satisfactory and are particularly poor for poly[(G-C)·(G-C)] in RNA geometry and poly(A·T) in B-DNA geometry. Attempts to improve agreement with measured spectra by varying monomer properties have been only partially successful for these calculations, but they illustrate the types of changes that may prove to be necessary. Calculations using other published X-ray coordinates for certain deoxypolynucleotides of simple sequence, some of which are quite different from B-DNA coordinates, did not result in better agreement with measured spectra. Finally, the dependence of the calculated CD on chain length is examined. Results show that non-nearest neighbor interactions can be important when runs of 3 or more identical base pairs appear in a given sequence.     

Interactions of Molecules with Nucleic Acids. X. Covalent Intercalat e Binding of the Carcinogenic BPDE I(+) to Kinked DNA

Abstract

A theoretical model is proposed for the covalent binding of (+) 7 β,8α-dihydroxy-9α, 10α- epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene denoted by BPDE I(+), to N2 on guanine. The DNA must kink a minimum of 39° to allow proper hybrid configurations about the C10 and N2 atoms involved in bond formation and to allow stacking of the pyrene moiety with the non-bonded adjacent base pair. Conservative (same sugar puckers and glycosidic angles as in B-DNA) and non-conservative (alternating sugar puckers as in intercalation sites) conformations are found and they are proposed structures in pathways connecting B-DNA, an intercalation site, and a kink site in the formation of a covalently intercalative bound adduct of BPDE I(+) to N2 on guanine. Stereographic projections are presented for (3′) and (5′) binding in the DNA. Experimental data for bending of DNA by BPDE, orientation of BPDE in DNA and unwinding of superhelical DNA is explained. The structure of a covalent intercalative complex is predicted to result from the reaction. Also, an anti ? syn transition of guanine results in a structure which allows the DNA to resume its overall B-form. The only change is that guanine has been rotated by 200° about its glycosidic bond so that the BPDE I(+) is bound in the major groove. The latter step may allow the DNA to be stored with an adduct which may produce an error in the genetic code.     

Interaction of parvalbumin of pike II with calcium and terbium ions

Fluorimetric titrations of parvalbumin II (pI 4.2) of pike (Pike II) with Ca2+ and Tb3+ show the CD and EF binding sites to be non-equivalent. The intrinsic binding constants of the strong and the weak sites obtained for Ca2+ are: KsCa = 1.6.10(8) M-1; KwCa = 6.6.10(5) M-1. Differences of the order of 100% were encountered between the Tb3+ binding constants obtained with four different versions of titration. Their average values are: KsTb = 1.9.10(11) M-1; KwTb = 1.0.10(7) M-1. The distances of the strong and the weak sites from the singular Tyr-48, rs = 9.5 A and r2 = 11.5 A, were derived from F?rster-type energy transfer and proved compatible with the X-ray structure of parvalbumin III (pI 4.2) of carp (CarpIII). From the distances, it is suggested that CD is the strong and EF the weak metal-binding site of PikeII. Tb3+ was shown by CD spectroscopy to have the same structural effect on PikeII as Ca2+. Removal of the metal ions from PikeII results in a decrease of helix content as monitored by CD spectroscopy. This decrease is larger than that in CarpIII. A concomitant decrease of the fluorescence quantum yield at nearly constant decay time is indicative of mainly static quenching, probably by the non-coordinating carboxylate groups. The maximum helix content is almost completely reestablished upon binding of the first metal ion. However, small changes of the energy transfer in PikeII with one terbium ion bound to the strong site indicate fine structural rearrangements of the strong binding site when Ca2+ is bound to the weak one.     

Grand canonical Monte Carlo molecular and thermodynamic predictions of ion effects on binding of an oligocation (L8+) to the center of DNA oligomers.

Grand canonical Monte Carlo (GCMC) simulations are reported for aqueous solutions containing excess univalent salt (activities a +/- = 1.76-12.3 mM) and one of the following species: an octacationic rod-like ligand, L8+; a B-DNA oligomer with N phosphate charges (8 < or = N < or = 100); or a complex resulting from the binding of L8+ at the center of an N-mer (24 < or = N < or = 250). Simplified models of these multiply charged species are used in the GCMC simulations to predict the fundamental coulombic contributions to the following experimentally relevant properties: 1) the axial distance over which ligand binding affects local counterion concentrations at the surface of the N-mer; 2) the dependence on N of GCMC preferential interaction coefficients, gamma 32MC identical to delta C3/delta C2l a +/-, T, where C3 and C2 are, respectively, the molar concentrations of salt and the multiply charged species (ligand, N-mer or complex); and 3) the dependence on N of SaKobs identical to d in Kobs/d in a +/- = delta (magnitude of ZJ + 2 gamma 32J), where Kobs is the equilibrium concentration quotient for the binding of L8+ to the center of an N-mer and delta denotes the stoichiometric combination of terms, each of which pertains to a reactant or product J having magnitude of ZJ charges. The participation of electrolyte ions in the ligand binding interaction is quantified by the magnitude of SaKobs, which reflects the net (stoichiometrically weighted) difference in the extent of thermodynamic binding of salt ions to the products and reactants. Results obtained here from GCMC simulations yield a picture of the salient molecular consequences of binding a cationic ligand, as well as thermodynamic predictions whose applicability can be tested experimentally. Formation of the central complex is predicted to cause a dramatic reduction in the surface counterion (e.g., Na+) concentration over a region including but extending well beyond the location of the ligand binding site. For binding a cationic ligand, SaKobs is predicted to be negative, indicating net electrolyte ion release in the binding process. At small enough N, -SaKobs is predicted to decrease strongly toward zero with decreasing N. At intermediate N, -SaKobs appears to exceed its limiting value as N-->infinity.     

Thermodynamics of thermal unfolding of bovine apo-alpha-lactalbumin

Thermal unfolding of bovine alpha-lactalbumin in 10 mM borate buffer at pH 8.0 in the presence of 0.01-1.0 M NaCl was studied in terms of CD ellipticity. The apoprotein changes the conformation from a native-like (N) to an unfolded (U) form, which has an appreciable amount of the secondary structure but no tertiary structure, in the two-state type. Various thermodynamic parameters of the transition were analyzed. The differences in enthalpy and heat capacity between the N and U states are similar to the corresponding differences of the holoprotein obtained with the calorimetric method by Pfeil. It is shown that one Na+ binds with a binding constant larger than 10(2)-10(3) M-1 to a specific site (probably to the Ca2+-binding site) in the molecule and the bound Na+ stabilizes the N form of the apoprotein.     

Specific 3H-DMCM binding to a non-benzodiazepine binding site after silver ion treatment of rat brain membranes

Specific binding of the BZ-receptor ligand 3H-DMCM to rat cortical membranes was dramatically enhanced by preincubation of the homogenate with 0.1 mM silver (Ag+) nitrate. The binding was completely inhibited by midazolam. Nevertheless, the pharmacological specificity of the Ag+-enhanced 3H-DMCM binding was different from that of BZ-receptors. Furthermore, the Bmax value, the regional distribution and the molecular target size determined by radiation inactivation analysis of the Ag+-enhanced binding site were different from those of BZ-receptors. The results indicate that Ag+-enhanced 3H-DMCM binding represent a high affinity metal complex formation between 3H-DMCM and an unknown brain specific protein of approximately 100,000 daltons molecular weight.     

Molecular dynamics investigation of the interaction between DNA and distamycin

The complex of the minor groove binding drug distamycin and the B-DNA oligomer d-(CGCAAATTTGCG) was investigated by molecular dynamics simulations. For this purpose, accurate atomic partial charges of distamycin were determined by extended quantum chemical calculations. The complex was simulated without water but with hydrated counterions. The oligomer without the drug was simulated in the same fashion and also with 1713 water molecules and sodium counterions. The simulations revealed that the binding of distamycin in the minor groove induces a stiffening of the DNA helix. The drug also prevents a transition from B-DNA to A-DNA that was found to occur rapidly (30 ps) in the segment without bound distamycin in a water-free environment but not in simulations including water. In other simulations, we investigated the relaxation processes after distamycin was moved from its preferred binding site, either radially or along the minor groove. Binding in the major groove was simulated as well and resulted in a bound configuration with the guanidinium end of distamycin close to two phosphate groups. We suggest that, in an aqueous environment, tight hydration shells covering the DNA backbone prevent such an arrangement and thus lead to distamycin's propensity for minor groove binding.     

Impact of calcium on N1 influenza neuraminidase dynamics and binding free energy

The highly pathogenic influenza strains H5N1 and H1N1 are currently treated with inhibitors of the viral surface protein neuraminidase (N1). Crystal structures of N1 indicate a conserved, high affinity calcium binding site located near the active site. The specific role of this calcium in the enzyme mechanism is unknown, though it has been shown to be important for enzymatic activity and thermostability. We report molecular dynamics (MD) simulations of calcium‐bound and calcium‐free N1 complexes with the inhibitor oseltamivir (marketed as the drug Tamiflu), independently using both the AMBER FF99SB and GROMOS96 force fields, to give structural insight into calcium stabilization of key framework residues. Y347, which demonstrates similar sampling patterns in the simulations of both force fields, is implicated as an important N1 residue that can “clamp” the ligand into a favorable binding pose. Free energy perturbation and thermodynamic integration calculations, using two different force fields, support the importance of Y347 and indicate a +3 to +5 kcal/mol change in the binding free energy of oseltamivir in the absence of calcium. With the important role of structure‐based drug design for neuraminidase inhibitors and the growing literature on emerging strains and subtypes, inclusion of this calcium for active site stability is particularly crucial for computational efforts such as homology modeling, virtual screening, and free energy methods. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Mechanism of copper mediated triple helix formation at neutral pH in Drosophila satellite repeats

The highly repeated Drosophila melanogaster AAGAGAG satellite sequence is present at each chromosome centromere of the fly. We demonstrate here how, under nearly physiological pH conditions, these sequences can form a pyrimidine triple helix containing T.A-T and CCu.G-C base triplets, stabilized by Cu2+ metal ions in amounts mirroring in vivo concentrations. Ultraviolet experiments were used to monitor the triple helix formation at pH 7.2 in presence of Cu2+ ions. Triplex melting is observed at 23 degrees C. Furthermore, a characteristic signature of triple helix formation was obtained by Fourier transform infrared spectroscopy. The stabilization of the C.G-C base triplets at pH 7.2 is shown to occur via interactions of Cu2+ ions on the third strand cytosine N3 atom and on the guanine N7 atom of the polypurine target strand forming CCu.G-C triplets. Under the same neutral pH conditions in absence of Cu2+ ions, the triple helix fails to form. Possible biological implications are discussed.     

Heterogeneous DNA binding modes of berenil.

Isothermal titration calorimetry (ITC) profiles of berenil bound to different DNAs show that, despite the strong preference of berenil for AT-rich regions in DNA, it can bind to other DNA sequences significantly. The ITC results were used to quantify the binding of berenil, and the thermodynamic profiles were obtained using natural DNAs as well as synthetic polynucleotides. ITC binding isotherms cannot be simply described when a single set of identical binding sites is considered, except for poly[d(A-T)2]. Ultraviolet melting of DNA and differential scanning calorimetry were also used to quantify several aspects of the binding of berenil to salmon testes DNA. We present evidence for secondary binding sites for berenil in DNA, corresponding to G+C rich sites. Berenil binding to poly[d(G-C)2] is also observed. Circular dichroism experiments showed that binding to GC-rich sites involves drug intercalation. Using a molecular modeling approach we demonstrate that intercalation of berenil into CpG steps is sterically feasible.     

Long-range allosteric effects on the B to Z equilibrium by daunomycin

Spectroscopic and fluorometric methods were used to study the binding of the anticancer drug daunomycin to poly[d(G-C)] and poly[d(G-m5C)] under a variety of solution conditions. Under high-salt conditions that favor the left-handed Z conformation, binding isotherms for the interaction of the drug with poly[d(G-C)] are sigmoidal, indicative of a cooperative binding process. Both the onset and extent of the cooperative binding are strongly dependent upon the ionic strength. The binding data may be explained by a model in which the drug preferentially binds to B-form DNA and acts as an allosteric effector on the B to Z equilibrium. At 2.4 M NaCl, binding of as little as one drug molecule per 20 base pairs (bp) results in the conversion of poly[d(G-C)] from the Z form entirely to the B form, as inferred from binding data and demonstrated directly by circular dichroism measurements. Similar results are obtained for poly[d(G-m5C)] in 50 mM NaCl and 1.25 mM MgCl2. Under these solution conditions, it is possible to demonstrate the Z to B structural transition in poly[d(G-m5C)] as a function of bound drug by the additional methods of sedimentation velocity and susceptibility to DNase I digestion. The transmission of allosteric effects over 20 bp is well beyond the range of the drug's binding site of 3 bp. Since daunomycin preferentially binds to alternating purine-pyrimidine sequences, which are the only sequences capable of the B to Z transition, the allosteric effects described here may be of importance toward understanding the mechanism by which the drug inhibits DNA replicative events.(ABSTRACT TRUNCATED AT 250 WORDS)     

A-DNA accommodates adducts derived from diol epoxides of polycyclic aromatic hydrocarbons bound in a "side-stacking" mode

The minor groove of undistorted A-DNA provides a good binding site for planar, hydrophobic moieties such as unmetabolized polycyclic aromatic hydrocarbons (PAHs), and the base pairs at the ends of short oligodeoxynucleotide helices. It also accommodates the chief adduct derived from the metabolically activated form of the carcinogen benzo[a]pyrene. B-DNA lacks such a site. Computerized models have been generated for the major (N2-guanine-linked) adducts formed at this site by both + and - enantiomers of anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (anti-BPDE) with poly(dG).poly(dC) in the A-DNA conformation. The BPDE adducts lie in the shallow, relatively hydrophobic minor groove of the A-DNA after empirical potential energy minimization using the program AMBER. We term this binding mode "side-stacking." The side-stacked + anti-BPDE may constitute the chief carcinogenic lesion derived from benzo[a]pyrene.     

A novel major groove binding site in B-form DNA for ethidium cation

A stably-bound external binding site for ethidium cation in the major groove of B-form DNA is proposed. This complex is stabilized by hydrogen bonding between this ligand and the nucleophilic centers O6 and N7 of guanine, both of which are accessible via the major groove. This binding site is not the same as the well-characterized electrostatically-stabilized external binding site, but rather is seen to be a covalently bound complex which is stabilized by two hydrogen bonds between the ethidium ligand and guanine in the double stranded (ds) B-form DNA. This site [(1), R. Monaco, F. Hasheer. J Biomol Struct Dyn 10, 675 (1993)] can only exist at very low occupancy ratios. The existence of this binding site leads directly to the expectation that there will exist particular mechanistic steps along the pathway of interaction between ethidium and ds B-DNA at low and high ligand concentrations that involve this binding mode. This would not only explain observations published recently [for example, see (2-6), W. Wilson, I. Lopp. Biopolymers 18, 3025 (1979); L. Wakelin, M. Waring. J Mol Biol 144, 183-214 (1980); A. Karpetyan, N. Mehrabian, G. Terzikian, A. Antonian, P. Vardevanian, M. Frank-Kamenetshii. Proceedings of the 10th Conversation, SUNY Albany, 275 (1998); P. Vardevanyan, A. Antonyan, G. Manukyan, A. Karapetyan. Experimental and Molecular Medicine 33, 205 (2001); P. Vardevanyan, A. Antonyan, L. Minasbekan, A. Karapetyan. Proceedings of the 2002 Miami Nature Biotechnology Winter Symposium, 2(S1), 144 (2002)] but also give insight into discrepancies reported in the literature over the years by different workers studying the mechanism of interaction between ethidium and DNA. In this paper this novel binding interaction is discussed, and it is shown how the elucidation of this interaction led to the proposal of two distinct mechanisms of intercalation between ds B-DNA and ethidium cation for high and low concentrations of ligand. Modeling studies show the stability, configuration, and relative energies of this outside binding site. It is expected that this externally bound complex between ethidium cation and ds B-form DNA will be experimentally detectable using fluorescent polarization and/or linear and circular dichroism spectroscopic studies [(7, 8) E. Tuite, U. Sehlstedt, P. Hagmar, B. Norden, M. Takahashi. Euro J Biochem 243, 482-492 (1997); T. Hard. Biopolymers 26, 613-618 (1987)].     

Effects of silver ion on the calcium-induced calcium release channel in isolated sarcoplasmic reticulum

Ag+-induced Ca2+ release in isolated sarcoplasmic reticulum (SR) was studied by the stopped flow method monitoring chlortetracycline fluorescence change. After improving the experimental procedure, the initial rate of Ca2+ release could be determined more precisely than before. Micromolar concentrations of Ag+ specifically enhanced Ca2+ efflux from heavy fraction of SR vesicles (HSR). This specific effect was referred to as Ag+-induced calcium release. The Ag+-induced Ca2+ efflux was activated by caffeine and ATP, but was inhibited by Mg2+ and procaine. Further, Ag+ enhanced the Ca2+-induced Ca2+ release over the whole range of Ca2+ concentrations, similarly to ATP. Parallel to Ca2+ efflux, Mg2+ efflux, measured by the same method, was also activated by Ag+. Choline permeability determined by the light scattering method was also activated by Ag+. The results suggest that Ag+ binds to the activation site of the Ca2+-induced Ca2+ release channel and opens the channel. The Ag+ binding site is different from the Ca2+ binding site but similar to the ATP binding site.     

Interaction of Ag+ ions with DNA and its monomers

Differential UV spectra of DNA and its monomers that were induced by Ag+ ions were measured, and the effect of ions on the parameters of the helix-coil transition was studied. The data obtained confirm the existence of "strong" and "weak" modes of binding of Ag+ to DNA. The earlier proposed proton transfer from N1G to N3C, which is determined by the interaction of Ag+ with N7G (a "strong" complex), follows immediately from the shape of the differential UV spectra. The positive cooperativity of the binding of Ag+ to DNA upon the formation of a "weak" complex is due to the cooperativity of the transition of DNA to a new double-helical conformation. A model of this conformation is proposed which suggests the formation of Hougsteen GC and AT pairs.     

The flexibility of the nucleic acids: (III). The interaction of an aliphatic diamine, putrescine, with flexible B-DNA

A theoretical modelling of the interaction of putrescine (H3+N-(CH2)4-(+NH3) with DNA is carried out, introducing two new features which make the simulation of this interaction considerably more realistic. Firstly, the DNA to which putrescine is bound is fully flexible and thus able to respond to the distorting influence of the ligand. Secondly, the effect of changing the ratio of DNA base pairs per bound ligand is explicitly modelled. In this way, we have been able to confirm the experimentally known preference of putrescine binding with AT base pairs in B-DNA, but we also show, through the new features introduced, that the nature of the binding site of the ligand and the resulting impact on DNA conformation is strongly modified by the ligand binding density.     

左手螺旋Z-DNA的研究 Ⅴ 环境因子对Oligo-d(G-C)_6构象的影响

脱氧寡聚核苷酸ｄ（Ｇ－Ｃ）６是迄今所报道的同类脱氧多聚核苷酸中能形成左手螺旋ＤＮＡ（Ｚ－ＤＮＡ）的最短序列。环境因子如ＰＨ,温度和离子的类别与浓度对ｄ（Ｇ－Ｃ）６在溶液中的Ｚ－构象的形成和Ｂ－、Ｚ－构象的相对稳定性有着明显的影响。在合适的条件下,ｄ（Ｇ－Ｃ）６在溶液中可呈现Ｂ－构象区,Ｚ－构象相对稳定区和Ｂ－、Ｚ－构象跃迁过渡区。