Binding of berberine to human telomeric quadruplex – spectroscopic, calorimetric and molecular modeling studies Amit Arora 1 , Chandramouli Balasubramanian 1 , Niti Kumar 1 , Saurabh Agrawal 1 , Rajendra P. Ojha 2 and Souvik Maiti 1 1 Proteomics and Structural Biology Unit, Institute of Genomics and Integrative Biology, CSIR, Delhi, India 2 Biophysics Unit, Department of Physics, DDU Gorakhpur University, India The telomere is a region of highly repetitive DNA at the end of a linear chromosome that protects the terminal ends of chromosomes from being recognized as damaged DNA and allows faithful chromosome replication during the cell cycle [1,2]. Mammalian telomeres consist of several kilobase pairs of double- stranded G-rich DNA and a 100–200 base single- stranded overhang on the 3¢-end [3,4]. A host of telomere-associated proteins, including TRF1, TRF2 and POT1, ensures that the telomeric single-stranded overhang does not trigger DNA damage response pathways or lead to abnormal chromosomal rearrange- ments [5–7]. Exposure of the 3¢-end due to uncapping results in cellular senescence and apoptosis [8,9]. Telo- merase, a ribonucleoprotein reverse transcriptase enzyme (composed of both RNA and proteins), uses its internal RNA component (complementary to the telomeric single-stranded overhang) as a template for synthesis of telomeric DNA A(GGGTTA) n , directly at the ends of chromosomes. Telomerase is present in most fetal tissues, normal adult male germ cells, inflammatory cells, proliferative cells of renewal tissues, and most tumor cells. Importantly, telomerase is active in a majority of human cancer cells but is inactive in most normal somatic cells [10]. It has been shown previously that formation of intramolecular G-quadruplexes by the telomeric G-rich strand inhibits the activity of telomerase [10]. Therefore, ligand- induced stabilization of intramolecular telomeric G-quadruplexes has become an attractive strategy for Keywords berberine; hydration; quadruplex; quadruplex–ligand interaction; thermodynamics Correspondence S. Maiti, Proteomics and Structural Biology Unit, Institute of Genomics and Integrative Biology, CSIR, Mall Road, Delhi 110 007, India Fax: +91 11 2766 7471 Tel: +91 11 2766 6156 E-mail: souvik@igib.res.in (Received 11 April 2008, revised 16 May 2008, accepted 9 June 2008) doi:10.1111/j.1742-4658.2008.06541.x This study examines the characteristics of binding of berberine to the human telomeric d[AG 3 (T 2 AG 3 ) 3 ] quadruplex. By employing UV-visible spectroscopy, fluorescence spectroscopy and isothermal titration calorime- try, we found that the binding affinity of berberine to the human telomeric quadruplex is 10 6 . The complete thermodynamic profile for berberine bind- ing to the quadruplex, at 25 °C, shows a small negative enthalpy (DH)of )1.7 kcalÆmol )1 , an entropy change with TDS of +6.5 kcalÆmol )1 , and an overall favorable free energy (DG)of)8.2 kcalÆmol )1 .Through the temper- ature dependence of DH, we obtained a heat capacity (DC p )of)94 (± 5) calÆmol )1 ÆK )1 . The osmotic stress method revealed that there is an uptake of 13 water molecules in the complex relative to the free reactants. Furthermore, the molecular modeling studies on different quadruplex– berberine complexes show that berberine stacking at the external G-quartet is mainly aided by the p–p interaction and the stabilization of the high negative charge density of O6 of guanines by the positively charged N7 of berberine. The theoretical heat capacity (DC p ) values for quadruplex– berberine models are )89 and )156 calÆmol )1 ÆK )1 . Abbreviations H 2 TMPyP 4 , 5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H,23H-porphine; ITC, isothermal titration calorimetry; MMPBSA, molecular mechanics Poisson–Bolzmann surface area; SASA, solvent-accessible surface area. FEBS Journal 275 (2008) 3971–3983 ª 2008 The Authors Journal compilation ª 2008 FEBS 3971 the development of anticancer ligands. A molecule that has (a) a p-delocalized system, (b) a partial positive charge in the center of the molecular scaffold and (c) positively charged substituents to interact with the grooves, loops and the negatively charged phosphate backbone is most likely to interact with, and thus stabilize, G-quadruplexes. A number of G-quadruplex- interacting agents with the above-mentioned features, typically porphyrins [11–16], anthraquinones [17], perylenes [18] and carbocyanines [19], have been developed and shown to promote and ⁄ or stabilize quadruplex structures. In past few years, Neidle and co-workers have reported a number of trisubstituted acridine analogs with a variety of side-chain modifi- cations and stereoisomer variations exhibiting strong G-quadruplex binding, high selectivity for quadruplex over duplex DNA, and associated telomerase inhibitory activity in the nanomolar range [20–22]. The rational design of new therapeutic agents that bind to quadruplexes in a structure-specific manner is of considerable interest and urgency. Many small molecules that bind to quadruplexes have proven to be effective therapeutic agents, although the exact mode of binding and nature of thermodynamic forces that regulate DNA–ligand interactions are often poorly understood. This limited knowledge hampers many efforts to rationally modify existing ligands and ⁄ or design new therapeutic agents that bind to target quadruplex structures with predictable affinity and specificity. Characterization of the forces that govern quadruplex–ligand interactions traditionally relies on detailed knowledge of the thermodynamic and struc- tural properties of the ligand, the DNA, and the com- plex. Berberine, an isoquinoline alkaloid from plants, is a planar molecule with an extended p-delocalized system having a partial positive charge on N7 [23] (Fig. 1). It has been shown that berberine and its analogs bind to telomeric G-quadruplex and inhibit the telomerase activity [24,25]. Studies show that these molecules have high selectivity for G-quadruplex over duplex DNA, and the aromatic moieties of the mole- cule play a dominant role in quadruplex binding, implying that this molecule could be an attractive scaf- fold to develop new ligands targeting G-quadruplex selectively. To obtain comprehensive knowledge on the interaction of this scaffold, we performed spectro- scopic, calorimetric and molecular modeling studies to obtain thermodynamic and structural details of the quadruplex–berberine interaction. Results Equilibrium binding studies by UV-visible spectroscopy To gain insight into the interaction between berberine and the G-quadruplex formed by the telomere, UV spectra of berberine in the absence and presence of quadruplex were obtained. The resulting absorption spectra are illustrated in Fig. 2. The UV spectra of berberine show two distinct peaks at 341 and 421 nm. Addition of increasing amounts of quadruplex results in hypochromicity (34–40%) and a moderate batho- chromic shift of 7 nm for the high-energy peak from 341 to 348 nm in the UV-visible spectra of berberine, and hyperchromicity and a red shift of 19 nm for the lower-energy peak from 421 to 440 nm, indicating Fig. 1. Chemical structure of berberine. Fig. 2. Absorbance spectra of 1 lM berberine in 50 mM MES buffer (pH 7.4) and 100 m M KCl in the absence and presence of succes- sive additions of quadruplex at 25 °C. The inset is the Scatchard plot of r ⁄ C versus r, where r is the ratio of bound berberine to the total base pair concentration, and C is the concentration of free ligand. Data were fitted to the McGhee–von Hippel neighbor exclu- sion model. Telomeric quadruplex–berberine interaction A. Arora et al. 3972 FEBS Journal 275 (2008) 3971–3983 ª 2008 The Authors Journal compilation ª 2008 FEBS interaction of berberine with quadruplex. The occur- rence of sharp isobestic points at 359, 383 and 445 nm clearly indicates the existence of equilibrium in the binding. Ligand binding with DNA through intercala- tion usually results in hypochromicity and bathochro- mism due to strong stacking interactions between an aromatic chromophore and the base pairs of DNA. These spectral characteristics suggest a mode of bind- ing that involves a stacking interaction between berber- ine and the quartet of quadruplex. The absorbance change at 341 nm of the berberine absorption spectra upon successive addition of quadruplex was used to construct a Scatchard plot. Analysis of this Scatchard plot yielded a binding affinity of (1.2 ± 0.2) · 10 6 m )1 and a binding site density of 0.9 at 25 °C. Equilibrium binding studies by the fluorescence method Fluorescence emission spectra for berberine in the absence and presence of different amounts of quadru- plex were recorded in order to study the binding event. Figure 3 shows the effect of successive addition of quadruplex on the fluorescence emission spectra of berberine. It is seen that increasing the concentration of quadruplex results in a gradual increase in the fluo- rescence intensity of berberine. The ratio of the fluo- rescence intensity of berberine in the presence and absence of quadruplex is about 50. The k max in the flu- orescence emission spectra shifts to the blue end by 5 nm. The spectral changes arise from the change in the environment of berberine, which reveals that berberine is binding with quadruplex. The change in fluorescence intensity at 522 nm due to addition of quadruplex solution was used to construct the binding isotherm (inset of Fig. 3). Analysis of this isotherm following 1 : 1 binding stoichiometry using Eqn (7) (see Experimental procedures) gives a binding affinity of (1.2 ± 0.1) · 10 6 m )1 at 25 °C. Thermodynamic parameters calculated for the quadruplex–berberine binding are presented in Table 1. Equilibrium binding studies by the isothermal titration calorimetry (ITC) method With recent advances in the sensitivity and reliability of the calorimeter, ITC has become an important tool for the direct measurement of thermodynamic para- meters in various biological interactions [26,27]. ITC yields thermodynamic parameters such as Gibbs free energy change (DG), enthalpy change (DH), and entropy change (DS), along with the number of bind- ing sites (n) in a single experiment. Also, determination of binding enthalpy as a function of temperature yields changes in heat capacity (DC p ) associated with an interaction that provides valuable insights into the type and magnitude of forces involved. Therefore, we have utilized ITC to characterize the thermodynamics of binding of berberine to quadruplex. Calorimetric titra- tions were performed at different temperatures to directly measure the binding enthalpy. Figure 4A shows a typical titration curve obtained at 25 °C. The area under the heat burst curves was determined by integration to yield the heat of injection associated with the reaction. These injection heats were corrected by subtraction of the corresponding dilution heats derived from the injection of identical amounts of ber- berine into the buffer alone. The corrected isotherms obtained at five different temperatures are shown in Fig. 4B. All related thermodynamic parameters are presented in Table 2. The binding affinity measured from ITC is (0.4 ± 0.1) · 10 6 m )1 at 25 °C. At all temperatures studied, the binding enthalpies were found to be negative, with their magnitude increasing Fig. 3. Fluorescence emission spectra of 0.5 lM berberine in 50 m M MES buffer (pH 7.4) and 100 mM KCl in the absence and presence of successive additions of quadruplex at 25 °C. The inset is the plot of DF versus quadruplex concentration. Data were fitted to Eqn (7) to extract the binding affinity (Experimental procedures). Table 1. Thermodynamic parameters obtained for quadruplex–ber- berine binding at 25 °C. K b is the binding constant determined from spectroscopic titrations in 50 m M MES buffer (pH 7.4), DG is the net binding free energy calculated using DG = )RT ln K b , and DH is the binding enthalpy determined directly by ITC and used to calcu- late the entropy change, using DG = DH)TDS. K b (·10 6 M )1 ) DG (kcalÆmol )1 ) DH (kcalÆmol )1 ) TDS (kcalÆmol )1 ) 1.2 ± 0.1 )8.2 ± 0.8 )1.7 ± 0.2 6.5 ± 0.7 A. Arora et al. Telomeric quadruplex–berberine interaction FEBS Journal 275 (2008) 3971–3983 ª 2008 The Authors Journal compilation ª 2008 FEBS 3973 with an increase in temperature. In all cases, the stoi- chiometry was found to be one mole of ligand binding per mole of quadruplex. Apparent discrepancy between spectroscopic and calorimetric binding constants Examination of Tables 1 and 2 shows that the K b val- ues determined from the spectroscopic and ITC data differ by one order at 25 °C, despite the use of identi- cal salt and buffer conditions. The binding constant can be determined accurately when titrant is added to a fixed and constant concentration [Q 0 ] of DNA, such that [Q 0 ] is in the range of 1 ⁄ K A . In UV and fluores- cence binding experiment, [Q 0 ]=1lm and 0.5 lm respectively, which is in the range of 1 ⁄ K A (1.2 lm). However, the ITC experiment was performed at [Q 0 ]=10lm, which is much larger than 1 ⁄ K A (1.2 lm). ITC could not be used in this case to obtain an accurate value for K b , as the low site concentration required would give a heat signal below the sensitivity of the instrument. For a quadruplex concentration of 1 lm in the cell, the heat output was not significantly greater than the heats of ligand dilution. If the dilution heats were small and the binding enthalpy was large, then it would be possible to obtain a binding isotherm using a quadruplex concentration of 0.5–1 lm. How- ever, these conditions are not met. Despite these prob- lems, ITC can still be used to accurately and directly measure the binding enthalpy and stoichiometry for this interaction. ITC remains an invaluable technique for determining binding enthalpies, even in cases where the binding constant cannot be determined accurately [28]. Heat capacity measurements Supplementary Fig. S1 shows the temperature depen- dence of all thermodynamic parameters. No curvature in the plots of thermodynamic constants versus tem- perature is apparent over this temperature range, and all of the plots are fitted with linear functions. The heat capacity change (DC p ) for a binding interaction Fig. 4. (A) Sample thermogram for the calorimetric titration of 100 l M berberine into 10 lM quadruplex at 25 °C. (B) Integrated heats (after subtraction of heat of dilution for berberine) versus ber- berine to quadruplex molar ratio plot at 10 °C(h), 15 °C(O), 20 °C (D), 25 °C(,) and 30 °C(e). The first data point was eliminated in the data fit. Table 2. Thermodynamic parameters obtained from ITC experiments for quadruplex–berberine binding in 50 mM MES buffer (pH 7.4) buffer containing 100 m M KCl. Thermodynamic parameters were obtained for berberine binding to the preformed telomeric quadruplex at 25 °C. The quadruplex concentration in the cell was 10 l M and the berberine concentration in the syringe was 100 lM. DG was determined using the relationship DG = )RT ln K b , where R is the universal gas constant, T is temperature, and K b is the binding affinity for the quadruplex– berberine interaction. DH and DS correspond to the enthalpy and entropy change for the binding, respectively, and DG corresponds to the free energy change of binding. DC p is the heat capacity change associated with the quadruplex–berberine interaction and is calculated using Eqn (1) as described in the text. Values determined from microcalorimetric data. Temperature (°C) DH (kcalÆmol )1 ) K b · 10 )6 (M )1 ) n DG (kcalÆmol )1 ) TDS (kcalÆmol )1 ) DC p (calÆmol )1 ÆK )1 ) 10 )0.2 (±0.2) 4.5 (±0.2) 0.85 )8.5 ± 0.9 8.3 ± 0.8 )94.0 (±5.0) 15 )0.9 (±0.2) 2.0 (±0.1) 0.90 )8.3 ± 0.8 7.4 ± 0.7 20 )1.3 (±0.3) 0.8 (±0.05) 0.95 )7.9 ± 0.8 6.6 ± 0.7 25 )1.7 (±0.3) 0.4 (±0.07) 1.00 )7.7 ± 0.8 6.0 ± 0.6 30 )2.1 (±0.3) 0.2 (±0.05) 0.90 )7.3 ± 0.7 5.2 ± 0.5 Telomeric quadruplex–berberine interaction A. Arora et al. 3974 FEBS Journal 275 (2008) 3971–3983 ª 2008 The Authors Journal compilation ª 2008 FEBS can be determined from the temperature dependence of the observed binding enthalpy using the standard relationship: DC p ¼ dDH cal =dT ð1Þ The slope of the resulting line of DH versus tempera- ture (T) in supplementary Fig. S1 yields DC p of )94 ± 5 calÆmol )1 ÆK )1 for the binding of berberine to quadruplex. Thus, berberine binding to quadruplex is associated with a negative heat capacity change that falls within a range that is frequently observed for both nucleic acid–ligand and protein–ligand inter- actions [29,30]. Hydration change due to the binding obtained by the osmotic stress method The osmotic stress method has been used extensively to evaluate the participation of water molecules in a wide variety of biochemical reactions [30]. Any equilib- rium that involves changes in the water molecules associated with a biopolymer is sensitive to changes in the water activity (a W ) [31–33]. Water activity can in turn be manipulated by the addition of low molecular weight cosolutes, which themselves do not interact with the biopolymer but are assumed to change the water activity. Equilibria that are coupled with hydra- tion changes are influenced by the osmolyte concentra- tion, as described by Qu & Chaires [33]: dlnðK s =K 0 Þ=d½Osm¼ÀDn w =55:6 ð2Þ where ln(K s ⁄ K 0 ) is the change in the binding free energy, [Osm] is the osmolality (moles of solute per kg of solvent) of the solution, and Dn w is the difference in the number of bound water molecules between the complex and the free reactants. The change in the binding affinity upon change in osmolyte concentration is shown in supplementary Fig. S2. As the concentra- tion of osmolyte increases, the affinity of berberine for the quadruplex binding site decreases. This observation is consistent with the acquisition of water by the com- plex relative to the DNA. Using Eqn (2), the average number of exchanged water molecules is found to be 13 ± 2. Molecular modeling studies Computational methods are widely used to investi- gate biomolecules and complexes, and have been shown to be valuable for a deeper understanding of the structural, dynamic and energetic properties. The mixed hybrid NMR structure of the human telomeric quadruplex was used for study (supplementary Fig. S3). The structure has two external G-quartets that can act individually as binding sites for berber- ine [25]. Berberine was docked against these external G-quartets, and the complexes were simulated in aqueous solution. The rmsd values of the heavy atoms of the whole complex (black) and without loop residues (gray) are shown in supplementary Fig. S4. The rmsd values of both the MH1 (5¢-end) and MH2 (3¢-end) complexes remain < 3 A ˚ . The rmsd values for the G-quartet (without loop residues) and berberine are conserved in both cases, and stay at < 1 A ˚ during the last 2 ns (inset in supplementary Fig. S4). The fluctuations of the loop residues are obvious, as they are not held tightly by hydrogen bonds and hence are free to move during dynamics. This observation has also been reported in previous studies on G-quadruplex structures [34,35]. The stacking of berberine over the external G-quartet is shown in Fig. 5 for the models. Berberine stacking over the G-quartet plane is aided by the formation of strong p–p aromatic stacking interactions between the berberine scaffold and the G-quartet plane. In addition, the positively charged nitrogen atom in berberine positions itself on the axis passing through the center of the G-quartet plane. Hence, a strong electrostatic inter- action can be expected between the positively charged nitrogen and the highly electron-rich central area of the G-quartet plane, due to the guanine carbonyl lone pairs. The positioning of the nitrogen atom was observed to be fairly retained in all models during dynamics. This suggests that the electrostatic interaction between the negatively charged clouds formed by O6 of guanines and the positively charged nitrogen atom plays an important role in this stabilization. The relative free energy components for the complex formation were estimated by the molecular mechanics Poisson–Bolzmann surface area (MMPBSA) approach. The calculations were performed on the basis of the single trajectories of the quadruplex–berberine complexes obtained from the explicit solvent simula- tions. The estimates are summarized in supplementary Table S1. Theoretical heat capacity calculation The heat capacity change upon complex formation is an informative measure that can provide insights into the exchange of water during the process. The relations connecting the changes in heat capacity to the burial of polar and nonpolar solvent-accessible surface area (SASA) during complex formation has been proposed A. Arora et al. Telomeric quadruplex–berberine interaction FEBS Journal 275 (2008) 3971–3983 ª 2008 The Authors Journal compilation ª 2008 FEBS 3975 and applied in a number of previous reports [29,36]. In our study, we used the relation proposed by Ren et al. [37], which is given as follows: DCp ¼ 0:382ðÆ 0:026ÞDA np À 0:121ðÆ 0:077ÞDA p ð3Þ Here, DA np and DA p represent the changes in SASA for nonpolar and polar groups, respectively. A sum- mary of the solvent-accessible areas and DC p values is shown in Table 3. The SASA is reduced upon complex formation, and the majority of the reduction was due to the burial of nonpolar surface. This is reflected in the negative values of the calculated heat capacity change. The calculated values are )89 and )156 calÆmol )1 ÆK )1 for MH1 and MH2 respectively. Discussion In order to understand biomolecule–ligand binding in terms of sequence-specific recognition and affinity, it is necessary to complement high-resolution structural data with accurate thermodynamic measurements. By using a combination of spectroscopic and ITC tech- niques, we have elucidated a complete thermodynamic profile (DG, DH, DS, K b , DC p and Dn w ) for the binding of berberine to the telomeric quadruplex. UV-visible absorption titration experiments show that the binding of berberine to G-quadruplexes results in a red shift (10–12 nm) and substantial hypochromicity (34–40%) in the k 341 nm of berberine. The red shift in the absorp- tion maxima and the observed hypochromicity of ber- berine in the presence of quadruplex may be interpreted in terms of stacking interactions between the quartet-forming guanine bases and the aromatic groups of berberine. Although the observed red shift is intermediate between what is observed for intercalation (> 15 nm) and for outside binding (< 8 nm) [38], the same extent of hypochromicity that is generally seen in the intercalated binding are observed, revealing that berberine interacts with quadruplex through stacking interactions between quartet and berberine, as happens in case of intercalative binding events. Recently, Wei Table 3. Summary of the changes in SASA in A ˚ 2 and heat capacity changes (DC p ) in calÆmol )1 ÆK )1 calculated for the complex models. DA tot is the change in total accessible surface area, DA np is the change in nonpolar SASA and DA p is the change in polar SASA. DC p is calculated using Eqn (3) as described in the text. Molecule DA tot (A ˚ 2 ) DA np (A ˚ 2 ) DA p (A ˚ 2 ) Calculated DC p (calÆmol )1 ÆK )1 ) MH1 4306 1533 2773 )89 Quadruplex 4248 1370 2879 Berberine 550 459 91 DA tot = )493 DA np = )296 DA p = )197 MH2 4079 1419 2659 )156 Quadruplex 4172 1424 2748 Berberine 551 461 90 DA tot = )645 DA np = )466 DA p = )179 A B G 20 G 2 G 8 G 16 G 4 G 10 G 14 G Fig. 5. Stacking of berberine on the G-quartet face A-MH1, B-MH2. The G-quartet is shown as a stick model. Berberine is shown as a ball and stick model. Telomeric quadruplex–berberine interaction A. Arora et al. 3976 FEBS Journal 275 (2008) 3971–3983 ª 2008 The Authors Journal compilation ª 2008 FEBS et al. [15] have studied the interaction of cationic porphyrin [5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H, 23H-porphine] (H 2 TMPyP 4 ) with three distinct G-quadruplex DNAs, parallel-stranded (TG 4 T) 4 , dimer-hairpin-folded (G 4 T 4 G 4 ) 2 , and monomer-folded AG 3 (T 2 AG 3 ) 3 , by UV resonance Raman spectroscopy, UV-visible absorption spectroscopy, fluorescence spec- troscopy, and surface-enhanced Raman spectroscopy. In their UV-visible absorption titration experiments, the same extent of red shift (11–13 nm) but larger hypochromicities (56–62%) were observed, indicating intercalative (containing the end stacking) binding of H 2 TMPyP 4 to these G-quadruplexes. Comparing these observations with ours, it can be concluded that berberine binds to quadruplex at external quartets through stacking interactions between the quartet- forming guanine bases and the aromatic groups of berberine. The lower red shift and moderate hypo- chromicity can be accounted for by the partial inter- calation by end stacking with the quartet. The 1 : 1 binding stoichiometry, as estimated from the present results, limits the interaction to a single binding mode. It was seen that addition of increasing concentrations of quadruplex results in a gradual increase in the fluorescence intensity of berberine, indicating the trans- fer of berberine from an aqueous environment to a hydrophobic environment. This observation rules out the possibility of outside stacking of berberine, where quenching of the chromophore fluorescence by solvent molecules could have been continued. In a recent study, Franceschin et al. [25], through molecular modeling, have shown that piperidino-berberine stacks on the terminal G-tetrad of the quadruplex. The sigmoidal binding isotherms obtained from ITC experiments are indicative of the existence of either a single binding site per quadruplex or a number of equivalent, but not necessarily independent, binding sites. At all temperatures, the stoichiometry for the quadruplex–berberine binding was found to be 1 : 1, confirming the results of our spectroscopic binding studies. The binding enthalpies were found to be neg- ative with increasing magnitudes upon increase in temperature. Berberine binds to quadruplex at 25 °C with a small, negative enthalpy (DH)of)1.7 kcalÆmol )1 and an entropy change with TDS of +6.5 kcalÆmol )1 with an overall favorable free energy (D G ) of )8.2 kcalÆmol )1 . The favorable binding of berberine comes from a combination of enthalpy and entropy terms that vary with temperature. The negative values of DH and positive values of DS are consistent with the characteristics of a combination of van der Waals, hydrophobic and electrostatic interactions in the binding process. The temperature dependence of the binding affinity was used to calculate the van’t Hoff enthalpy, which did not match the calorimetric enthalpy. The obtained van’t Hoff enthalpy was )12 kcalÆmol )1 , giving DH vH ⁄ DH cal ratios in excess of 1. A large difference between the van’t Hoff and calo- rimetric enthalpies could be due to substantial temper- ature-dependent behavior of associated reactions. This might originate from changes in hydrophobic hydra- tion [39], in which release of water molecules from hydrophobic surfaces upon binding results in loss of enthalpy (due to stronger hydrogen bonds of struc- tured water) and gain in entropy, a phenomenon known to be temperature-dependent [40–42]. Measure- ment of heat capacity changes (DC p ) associated with ligand–macromolecule binding can help to differentiate the nature of hydration changes, i.e. hydrophobic ver- sus polar hydration [43]. Unlike other thermodynamic parameters, which have contributions from various sources, DC p is believed to arise purely from molecular hydration associated with binding [44]. This parameter can thus be utilized to estimate the extent of burial or exposure of polar and nonpolar groups to bulk water upon molecular binding. The obtained DC p for the quadruplex–berberine interaction was )94 ± 5 calÆmol )1 ÆK )1 . In the case of intercalative as well as minor groove binding ligands, it was shown that DC p varies from )100 to )400 calÆmol )1 ÆK )1 [29,44]. How- ever, the large negative heat capacity change is highly correlated with hydration heat capacity changes that arise from burial of the hydrophobic area. As we have observed in our UV binding experiment, berberine binds to quadruplex by stacking on the terminal G-tet- rad of the quadruplex, so burial of the hydrophobic group is not extensive enough to show a sufficiently negative heat capacity change upon interaction. The free energy (DG hyd ) for the hydrophobic transfer of a ligand from aqueous solution to its macromolecular binding pocket is a function of the DC p for the binding reaction, and is given by the following relationship [44]: DG hyd ¼ 80  DC p ð4Þ According to this relationship, negative heat capacity changes result in a large negative (favorable) DG hyd value, which, in turn, shows a significant driving force for complex formation. As an illustrative example, the Chaires [37] and Wilson [29,45] groups applied Eqn (3) to show that the major driving force for the binding of various heterocyclic ligands (e.g. Hoechst 33258 and penta-amidine) to the minor groove of duplex DNA stems from the hydrophobic transfer of the ligands from solution to the DNA-binding site. A. Arora et al. Telomeric quadruplex–berberine interaction FEBS Journal 275 (2008) 3971–3983 ª 2008 The Authors Journal compilation ª 2008 FEBS 3977 Careful inspection of Table 1 clearly shows that the binding of berberine to the human telomeric quadru- plex having a small negative value of DH and a large positive value of TDS is predominantly entropically driven. Recently, Chaires has analyzed the binding data for 26 DNA–ligand interactions and discussed distinctive thermodynamic signatures for groove bind- ing and intercalation [46]. Groove-binding interactions are largely entropically driven, whereas intercalation reactions are driven by large favorable enthalpy con- tributions and are opposed by entropy. In the present study, the observed low negative enthalpy and high positive entropy change could constitute further evi- dence in support of stacking with the terminal quartet rather than intercalation. Rigidifying the DNA ought to exert an entropic cost, and this is the most likely explanation for the unfavorable entropy associated with intercalation [47]. In contrast, terminal stacking in the quadruplex structures should not rigidify the quadruplex structure, thus reducing the entropic cost as compared to intercalation. The origin of the favor- able entropic term in the present study is not also apparent. It has been argued that groove binding shows the favorable entropy because of the release of water molecules upon complex formation, by dis- placement of the ‘spine of hydration’ within the minor groove [48]. However, the osmotic stress study shows that the quadruplex–berberine complex acquires 13 molecules of water on average per mole of complex at 25 °C. Similar kinds of observation have been reported recently by Kiser et al. [49], where uptake of water molecules as well as a positive entropy change were observed when Hoechst, a groove binder, bound to oligomeric DNA. This dis- crepancy may be due to the inefficiency of the osmo- tic stress method in measuring the overall hydration change, as mentioned by Chaires [46] as well as by Kiser et al. [49]. The simulated structures show that the planar ber- berine molecule is stacked onto the G-quartet, posi- tioning the N7 positive charge above the center of the G-tetrad in the region of high negative charge density generated by the carbonyl groups to stabilize the complex via favorable p-stacked interactions between aromatic residues without significant disruption of the guanine tetrads. The binding enthalpy (DH) for the quadruplex–berberine complexes originates from the combination of polar solvation energy and favorable solute electrostatic, van der Waals and nonpolar sol- vation energy. The conformational entropy for the complexes arises from the loss of translational and rotational degrees of freedom. The overall free energy change (DG) for the quadruplex–berberine binding is found to be more favorable for the MH2 berberine– quadruplex complex (supplementary Table S1). Fur- thermore, the calculated DC p is highly negative and arises due to the loss of nonpolar SASA, whereas the accompanying uptake of water is associated with gain of polar SASA. This is because berberine is nonpolar in nature, and hence the more nonpolar accessible sur- face gets buried upon interacting with the quadruplex. On the other hand, the positive change in the polar surface area (DA p ) confirms that there is exposure of the polar surface on complex formation, further imply- ing the uptake of water molecules within the hydration layer. Conclusion The single-stranded G-rich telomeric 3¢-overhang at the ends of chromosomes can form unique secondary DNA structures, such as G-quadruplexes, which are known to inhibit telomerase activity and have thus become attractive targets for new anticancer ligands. However, a structure-based approach needs to be developed to design a new generation of binding agents that can selectively target such unique second- ary DNA structures. Ultimately, a comprehensive understanding of the thermodynamic and structural parameters of quadruplex–ligand complexes would aid in the design of new quadruplex-selective molecules and help to rationalize their in vivo performance. In this study, we have obtained comprehensive data on the thermodynamic and structural parameters involved in the quadruplex–ligand interaction, using a well-characterized human telomeric quadruplex and an alkaloid, berberine. It has been observed that bind- ing of berberine to the human telomeric quadruplex is associated with a small, negative enthalpy (DH) of )1.7 kcalÆmol )1 , a favorable free energy (DG) of )8.2 kcalÆmol )1 and a favorable entropy with TDS value of +6.5 kcalÆmol )1 ÆK )1 at 25 °C. A negative heat capacity change was observed when it was calcu- lated using two independent methods experimentally, from the temperature dependence of DH values, and theoretically based on surface area calculations. The theoretical value is more negative than the experimen- tal value. There was an uptake of 13 water molecules on average per complex, which provides an unfavor- able contribution to the free energy of the binding. Structural studies of the complex obtained from mole- cular dynamic studies reveal that berberine stacks over the G-tetrad, allowing overlap of the p-system of berberine primarily with two bases of each G-tetrad. The partial positive charge on the berberine N7 appears to act as a ‘pseudo’ potassium ion, and is Telomeric quadruplex–berberine interaction A. Arora et al. 3978 FEBS Journal 275 (2008) 3971–3983 ª 2008 The Authors Journal compilation ª 2008 FEBS positioned above the center of the G-tetrad in the region of high negative charge density generated by the carbonyl groups. Extension of this study to other known and well-established ligands, such as porphyrin and telomestatin, will be reported in due course. Experimental procedures Berberine chloride was obtained from Sigma and was used without any further purification. The 22-mer oligonucleo- tide from the telomere end, d(AGGGTTAGGGTTAGG GTTAGGG), was obtained from Sigma Genosys USA. Concentrations of oligonucleotide solutions were deter- mined from the absorbance at 260 nm, using the molar extinction coefficient for the G-rich strand, calculated by extrapolation of tabulated values of the dimers and mono- mer bases [50] at 25 °C, using procedures reported earlier [51]. All other reagents were of analytical grade. Milli Q water was used throughout all the experiments. All experiments were performed in 50 mm MES buffer (pH 7.4) containing 100 mm KCl at 25 °C, unless otherwise specified. UV-visible and fluorescence spectroscopy Quadruplex–berberine binding constants were determined by UV fluorescence, and Cary 400 (Varian) and Fluoro- max 4 (Spex) instruments were used for UV and fluores- cence titration experiments respectively. A fixed berberine concentration was titrated by increasing the quadruplex concentration in 50 mm MES buffer (pH 7.4) containing 100 mm KCl. Data were transformed into a Scatchard plot of r ⁄ C versus r, where r is the ratio of bound berberine to the total quadruplex concentration, and C f is the concen- tration of free ligand. In the Scatchard equation, r ⁄ C f = K b (n)r), where r is the number of moles of berberine bound to 1 mol of quadruplex (C b ⁄ C qua ), n is the number of equivalent binding sites, and K b is the affinity of ligand for those sites [52]. Data were fitted to the McGhee– von Hippel neighbor exclusion model. To determine the affinity of binding between berberine and quadruplex, fluorescence experiments were carried out at 25 °C using a fixed concentration (500 nm) of berberine and varying the quadruplex concentration (0–5 lm). For analysis of data, the observed fluorescence intensity was considered as the sum of the weighted contributions from free berberine and berberine bound to quadruplex: F ¼ð1 À a b ÞF 0 þ a b F b ð5Þ where F is the observed fluorescence intensity at each titrant concentration, F 0 and F b are the respective fluores- cence intensities of the initial and final states of titration, and a b is the mole fraction of berberine in bound form. Assuming 1 : 1 stoichiometry for the interaction as observed in the Scatchard plot, it can be shown that: ½L 0 a 2 b Àð½L 0 þ½Qþ1=K b Þa b þ½Q¼0 ð6Þ where K b is the binding constant, [L] 0 is the total berberine concentration, and [Q] is the added quadruplex concentra- tion. From Eqns (4,5), it can be shown that: DF ¼ðDF max =2½L 0 Þð½L 0 þ½Qþ1=K A ÞÀ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð½L 0 þ½Qþ1=K A Þ 2 À 4½L 0 ½Q q  ð7Þ where DF = F)F 0 and DF max = F max )F 0 , and F and F 0 are the initial and subsequent fluorescence intensities of the berberine at 522 nm, upon quadruplex addition. ITC ITC measurements were carried out on a VP-ITC titration calorimeter (MicroCal, Northampton, MA, USA). Before loading, the solutions were thoroughly degassed. The refer- ence cell was filled with the respective degassed buffer. The preformed quadruplex concentration (10 l m) was kept in the sample cell, and berberine (100 lm) in the same buffer was placed in a syringe of volume 300 lL. The berberine solution was added sequentially in 10 lL aliquots (for a total of 25 injections, 20 s duration each) at 4 min intervals. Sequential titrations were performed to ensure full occu- pancy of the binding sites by loading and titrating with the same ligand without removing the samples from the cell until the titration signal was essentially constant. The heats of dilution were determined in parallel experiments by injecting a berberine solution of the same concentration in the same buffer. The respective heat of dilution is sub- tracted from the corresponding binding isotherm prior to curve fitting. origin 5.0 software was used to fit the thermodynamic parameters to the heat profiles. Molecular modeling Literature studies based on NMR data reveal that the 22- mer human telomeric sequence assumes multiple intercon- vertible conformations, comprising the parallel, antiparallel and hybrid-type G-quadruplexes, in the presence of potas- sium ions [53]. However, no solution structure obtained in the presence of potassium ions is available for the 22-mer human telomeric sequence. Furthermore, the telomeric quadruplex adopts a mixed hybrid conformation in the pres- ence of berberine (1 : 1 molar ratio of berberine: telomeric quadruplex), as shown in the CD spectra (supplementary Fig. S5). We chose the hybrid-type NMR structure (Protein Data Bank ID 2hy9) [54] of the human telomeric quadru- plex, as shown in supplementary Fig. S3. The 2hy9 is a 26- mer mixed-hybrid structure of the human telomeric sequence in the presence of potassium ions. The four adenine residues, two from each terminal end, were removed for comparison A. Arora et al. Telomeric quadruplex–berberine interaction FEBS Journal 275 (2008) 3971–3983 ª 2008 The Authors Journal compilation ª 2008 FEBS 3979 purposes. The initial 3D coordinate for berberine was extracted from the crystal structure of the transcriptional receptor QacR from the Protein Data Bank (1jum) [55]. The extracted structure was then optimized, and the partial charges were derived with the HF ⁄ 6-31G* basis set in gaussian 03 [56]; this was followed by restrained electro- static potential calculation in the antechamber module of amber8. The remaining parameters for berberine were taken from the GAFF forcefield in amber8 [57]. The NMR structures possess two external G-quartets, shown in supple- mentary Fig. S3, that can independently act as binding sites for berberine stacking. The optimized structure of berberine was docked against the binding sites by defining the external G-quartet as the active site using the suflexdock module in sybyl 7.3 [58]. This resulted in two quadruplex– berberine complex models, referred to as MH1 and MH2. surflexdock provides multiple docked conformations, and the one with the lowest docking energy was considered as the starting structure for further simulation experiments. Molecular dynamics Two potassium ions were manually placed in the central channel between the G-quartet planes in the complex mod- els. The complex models were simulated using the amber8 [59] suite of programs with the Cornell et al. all-atom force field ff99 [60]. The complexes were neutralized with potas- sium ions, with the two inner ions retained inside. The sys- tems were then immersed in a periodic box of TIP3P water model, which extended approximately 8 A ˚ (in each direc- tion) from the solute in a truncated octahedron unit cell [61]. Simulations were performed with periodic boundary conditions, and the particle-mesh Ewald method was used to treat long-range electrostatics [62]. Bond lengths involving bonds to hydrogen atoms were constrained using shake [63]. A time step of 2 fs was used except for the equilibration phase, which was 1 fs. The direct-space cutoff used was 10 A ˚ . Simulations were performed at a constant temperature of 300 K. The Langevin coupling with a collision frequency of 1.0 was used for temperature regula- tion [64]. A constant pressure of 1 atm with isotropic mole- cule-based scaling with a relaxation time of 1 ps was used. The equilibration step involves multiple optimization and relaxation of the solvent and potassium ions in the bulk solvent with the solute and the two inner potassium ions fixed with restraints that include 500, 100, 250, 50, 100, 25, 50 and 10 kcalÆmol )1 A ˚ respectively. Then, the whole sys- tem was heated from 0 to 300 K at constant volume, and this was followed by equilibration for 25 ps at a constant temperature of 300 K and a pressure of 1 atm. The produc- tion phase was started at this stage and continued for 5 ns. All simulations were performed in an SGI Altix 450 cluster. The conformations in the trajectories were collected at intervals of 2 ps. Trajectory analyses were done using the ptraj program in amber 8. SASA calculation The SASA calculation was done using grasp 1.3 [65]. The lowest-energy structure evolved during simulation was used for the SASA calculation of the complexes. Surfaces for carbon, carbon-bound hydrogen and phosphorus are defined as nonpolar, and the remaining hydrophilic atoms are defined as polar. The grasp radii set was used in the calculation. Thermodynamics calculation The free energy estimates were performed by the MMPBSA approach, where the total free energy of binding is expressed as the sum of the contributions from the gas phase and solvation energies plus an additional term for the solute entropic contribution. This can be expressed in the following equation: G ¼ E gas þ E solv þ TS solute ð8Þ where E gas is the total gas-phase energy, E solv is the total solvation energy (polar + nonpolar), and TS solute corre- sponds to solute entropic effects. The analysis was done for the last 2 ns trajectory of the complexes. The snapshots for quadruplex and berberine were extracted from the complex trajectories at intervals of 10 ps. This yielded 100 snapshots in total. All counterions (except the two spanning the cen- tral channel of the G-quartet planes) and water molecules were stripped out from the trajectory prior to the thermo- dynamic analysis. The gas-phase energies of the solutes were calculated using the Cornell et al. force field [60] with no cutoff. Solvation free energies were computed as the sum of polar and nonpolar contributions using a contin- uum solvent representation. The polar contribution was calculated with the pbsa program in amber8. The dielectric constants used for the solute and the surrounding solvent were 1 and 80, respec- tively. The Cornell et al. radii set was used to define atom-centered spheres for the solute atoms, and a probe radius of 1.4 A ˚ was used for the solvent to define the dielectric boundary around the molecular surface. A lat- tice spacing of two grid points per A ˚ was used, and 1000 finite difference iterations were performed, excluding salt effect. The nonpolar solvent contribution was estimated from an SASA-dependent term, DE snp = c. SASA + b, where c was set to 0.0075 kcalÆA ˚ )2 and b to 0. The cal- culation for solute entropic contribution was performed with the nmode module in amber8. The snapshots were minimized in the gas phase using the conjugate gradient method for 5000 steps, using a distance-dependent dielec- tric of 4r (r is the interatomic distance) and with a con- vergence criterion of 0.1 kcalÆ(molÆA ˚ ) )1 for the energy gradient. The frequencies of the vibrational modes were computed for these minimized structures at 300 K, using normal mode analysis methodology. The thermodynamic Telomeric quadruplex–berberine interaction A. 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Binding of berberine to human telomeric quadruplex – spectroscopic, calorimetric and molecular modeling studies Amit Arora 1 , Chandramouli. performed spectro- scopic, calorimetric and molecular modeling studies to obtain thermodynamic and structural details of the quadruplex berberine interaction. Results Equilibrium