A circular dichroism study of DNA.platinum complexes. Differentiation between monofunctional, cis-bidentate and trans-bidentate platinum fixation on a series of DNAs

The circular dichroism (CD) spectra of a series of DNA . platinum complexes are presented. The following platinum compounds, [Pt(dien)Cl]Cl, cis-Pt(NH3)2Cl2, cis-Pt(en)Cl2, trans-Pt-(NH3)2Cl2, K[Pt(NH3)Cl3] and K2[PtCl4] were complexed with the DNA extracted from bacteria Micrococcus lysodeikticus (72% dG + dC), Escherichia coli (50% dG + dC), Clostridium perfringens (32% dG + dC) and salmon sperm (41% dG + dC). Strong differences were found between the different DNA . Pt complexes. Three types of spectra clearly demonstrate the different platinum binding modes on DNA. In the first type, the platinum compound, i.e. [Pt(dien)Cl]Cl, is fixed to DNA with only one bond (monofunctional complex formation) and no significant change of the CD positive band of DNA is found. The main feature of the second type is a continuous intensity decrease of the positive band as observed for trans-Pt(NH3)2Cl2 (trans-bidentate complex formation). The third type concerns the cis-bidentate platinum fixation obtained with cis-Pt(NH3)2Cl2, cis-Pt(en)Cl2, K[Pt(NH3)Cl3] and K2[PtCl4]. The CD spectra are in this case characterized by an increase in the positive Cotton effect which is dG + dC-dependent up to an rb value around 0.10 (where rb = number of platinum atoms bound per nucleotide), followed by a decrease until DNA saturation with platinum is reached. A linear decrease in the amplitude of the negative band is detected in all the complexes except in the case of the monofunctional DNA . Pt complexes. For the cis-bidentate and trans-bidentate platinum fixation, a continuous bathochromic shift occurs.     

Mutual Protein-Ligand Conformational Selection Drives cGMP vs. cAMP Selectivity in Protein Kinase G

Protein kinase G (PKG) is a major receptor of cGMP, and controls signaling pathways distinct from those regulated by cAMP. However, the contributions of the two substituents that differentiate cGMP from cAMP (i.e. 6-oxo and 2-NH₂) to the cGMP-versus-cAMP selectivity of PKG remain unclear. Here, using NMR to map how binding affinity and dynamics of the protein and ligand vary along a ligand double-substitution cycle, we show that the contributions of the two substituents to binding affinity are surprisingly non-additive. Such non-additivity stems primarily from mutual protein–ligand conformational selection, whereby not only does the ligand select for a preferred protein conformation upon binding, but also, the protein selects for a preferred ligand conformation. The 6-oxo substituent mainly controls the conformational equilibrium of the bound protein, while the 2-NH₂ substituent primarily controls the conformational equilibrium of the unbound ligand (i.e. syn versus anti). Therefore, understanding the conformational dynamics of both the protein and ligand is essential to explain the cGMP-versus-cAMP selectivity of PKG.     

A theoretical model for the binding of cis-Pt(NH3)2(+2) to DNA

The binding of cis-Pt(NH3)2B1B2 to the bases B1 and B2, i.e., guanine (G), cytosine (C), adenine (A), and thymine (T), of DNA is studied theoretically. The components of the binding are analyzed and a model structure is proposed for the intrastrand binding to the dB1pdB2 sequence of a kinked double helical DNA. Quantum mechanical calculations of the ligand binding energy indicates that cis-Pt(NH3)2(+2) (cis-PDA) binds to N7(G), N3(C), O2(C), O6(G), N3(A), N7(A), O4(T) and O2(T) in order of decreasing binding energy. Conformational analysis provides structures of kinked DNA in which adjacent bases chelate to cis-PDA. Only bending toward the major groove allows the construction of acceptable square planar complexes. Examples are presented for kinks of -70 degrees and -40 degrees at the receptor site to orient the base pairs for ligand binding to B1 and B2 to form a nearly square planar complex. The energies for complex formation of cis-PDA to the various intra-strand base sites in double stranded DNA are estimated. At least 32 kcal/mole separates the energetically favorable dGpdG.cis-PDA chelate from the dCpdG.cis-PDA chelate. All other possible chelate structures are much higher in energy which correlates with their lack of observation in competition with the preferred dGpdG chelate. The second most favorable ligand energy occurs with N3(C). A novel binding site involving dC(N3)pdG(N7) is examined. Denaturation can result in an anti----syn rotation of C about its glycosidic bond to place N3(C) in the major groove for intrastrand binding in duplex DNA. This novel intrastrand dCpdG complex and the most favored dGpdG structure are illustrated with stereographic projections.     

Studies on the interaction between antitrypanosome cis-DDP analogs with DNA

The nature of the conformational changes produced in DNA by cis-DDP analogs has been studied by physiochemical techniques. The UV spectra showed that the DNA undergoes bathochromic shifts accompanied by hyperchromic effects in reaction with specific analogs (cis-Pt(DDH) (mucobromic)2, cis-Pt(tranilcypromine)2Cl2 and cis-Pt(DDH) Cl2), while a different series of analogs (cis-Pt(DDH) (metafluorobenzoic)2 and cis-Pt(pentamidine)Cl2) induce a significative decrease in the absorbance at 258 nm. Moreover one of these analogs (cis-Pt(pentamidine)Cl2) causes strong stabilization of the double helix to heat denaturation. The CD spectra indicate moreover that cis-Pt(pentamidine)Cl2 modifies the secondary structure of the DNA in a significant way with an increase of the positive band and a decrease of the ellipticity of the negative band. The antitrypanosome activity of cis-Pt(pentamidine)Cl2 is probably due to inhibition of the intracellular parasites division in parasitized cells.     

Influence of substituent modifications on DNA binding energetics of acridine-based anticancer agents

The DNA binding energetics of a series of analogues derived from the anticancer agent N-[2-(dimethylamino)ethyl]-9-aminoacridine-4-carboxamide (AAC) are investigated. The effects of substituent modification at the C5 position of the acridine chromophore on the interaction of AAC with DNA are determined using spectrophotometry and isothermal titration calorimetry (ITC). The binding affinity and binding free energy associated with the interaction of AAC with DNA are significantly enhanced upon substitution at the C5 position. Energetic profiles describing ligand-DNA complex formation obtained from ITC indicate that C5 substitution significantly enhances binding enthalpy relative to the parent AAC. In many cases, the enhanced binding enthalpies of the C5-substituted analogues correlate with anticancer activity. Because of the cationic character of AAC and its analogues, the DNA binding properties of these compounds are dependent on ionic strength. To quantitate the ionic contributions to complex formation, the observed binding free energy of each compound is parsed into its polyelectrolyte and nonelectrostatic components. Enhanced nonelectrostatic contributions to the overall binding free energies observed with C5-substituted analogues relative to the parent AAC suggest that C5 substituents play a critical role in directing both thermodynamic mechanisms associated with complex formation and molecular interactions between the ligand and its DNA binding site. These studies have demonstrated that substitution of AAC at the C5 position results in enhanced DNA binding affinity and energetics.     

Ligand effects on the binding of cis- and trans-[PtCl2Am1Am2] to proteins

As part of a systematic study of the basic principles that govern the formation and reactivity of Pt-protein adducts, we report the effect of substituting the amine ligand of cis- and trans-[PtCl(2)(NH(3))(2)] complexes with bulkier planar aromatic or nonplanar cyclic amine ligands on the binding properties of the complexes to ubiquitin and to horse heart myoglobin. The ligand replacement had a different effect on the cis or trans isomers investigated. In the cis-Pt complexes, replacing one or both amine ligands by piperidine or 4-picoline dramatically decreased the binding of the complexes to the proteins studied, whereas in the substituted trans-Pt complexes replacement of the amine by a piperidine or 4-picoline increased the binding rate. This behavior may have to do with the different preferred binding sites of the cis- and trans-Pt complexes. The bulkier cis- or trans-Pt complexes investigated also did not display a preference for Met1 of ubiquitin, possibly owing to steric constraints imposed by the substituted ligands. The introduction of a charged piperazine ligand significantly decreased the rate of binding to the protein, possibly owing to electrostatic interactions or hydrogen-bond formations with the surface of the protein. The binding of the complexes to ubiquitin and myoglobin does not disrupt the folding of the proteins as judged by electrospray ionization mass spectrometry.     

Effect of biologically active compounds of divalent platinum on the properties of the liquid crystal "microphase" of DNA

The optical properties of the "microphases" modeling the state of the DNA molecule in the cell and formed of both the low molecular DNA and the DNA complexes with cis- and trans-isomers of dichlorodiamine platininum (II) were studied. It was shown that the intensive band characteristic of the circular dichroism spectrum of the initial DNA "microphase" was decreasing with binding of DNA to cis-Pt (II) or trans-Pt (II). The effect of cis-Pt (II) on the "microphase" optical properties was more significant than that of trans-Pt (II). The effect correlated with the biological activity of the cis- and trans-compounds of platinum. Possible causes of the decrease in the optical activity of the DNA "microphase" are discussed.     

Docking multiple conformations of a flexible ligand into a protein binding site using NMR restraints

A method is described for docking a large, flexible ligand using intra-ligand conformational restraints from exchange-transferred NOE (etNOE) data. Numerous conformations of the ligand are generated in isolation, and a subset of representative conformations is selected. A crude model of the protein-ligand complex is used as a template for overlaying the selected ligand structures, and each complex is conformationally relaxed by molecular mechanics to optimize the interaction. Finally, the complexes were assessed for structural quality. Alternative approaches are described for the three steps of the method: generation of the initial docking template; selection of a subset of ligand conformations; and conformational sampling of the complex. The template is generated either by manual docking using interactive graphics or by a computational grid-based search of the binding site. A subset of conformations from the total number of peptides calculated in isolation is selected based on either low energy and satisfaction of the etNOE restraints, or a cluster analysis of the full set. To optimize the interactions in the complex, either a restrained Monte Carlo-energy minimization (MCM) protocol or a restrained simulated annealing (SA) protocol were used. This work produced 53 initial complexes of which 8 were assessed in detail. With the etNOE conformational restraints, all of the approaches provide reasonable models. The grid-based approach to generate an initial docking template allows a large volume to be sampled, and as a result, two distinct binding modes were identified for a fifteen-residue peptide binding to an enzyme active site.     

Conformational selection or induced fit? A critical appraisal of the kinetic mechanism

For almost five decades, two competing mechanisms of ligand recognition, conformational selection and induced fit, have dominated our interpretation of ligand binding in biological macromolecules. When binding-dissociation events are fast compared to conformational transitions, the rate of approach to equilibrium, k(obs), becomes diagnostic of conformational selection or induced fit based on whether it decreases or increases, respectively, with the ligand concentration, [L]. However, this simple conclusion based on the rapid equilibrium approximation is not valid in general. Here we show that conformational selection is associated with a rich repertoire of kinetic properties, with k(obs) decreasing or increasing with [L] depending on the relative magnitude of the rate of ligand dissociation, k(off), and the rate of conformational isomerization, k(r). We prove that, even for the simplest two-step mechanism of ligand binding, a decrease in k(obs) with [L] is unequivocal evidence of conformational selection, but an increase in k(obs) with [L] is not unequivocal evidence of induced fit. Ligand binding to glucokinase, thrombin, and its precursor prethrombin-2 are used as relevant examples. We conclude that conformational selection as a mechanism for a ligand binding to its target may be far more common than currently believed.     

Synthesis and DNA threading properties of quaternary ammonium [Ru(phen)2(dppz)] derivatives

Ruthenium complexes with one dipyrido[3,2-a:2′-3′-c]phenazine (dppz) ligand, e.g. [Ru(phen)₂(dppz)]²⁺ (phen = phenanthroline), shows strong binding to double helical DNA and are well-known DNA “light-switch” molecules. We have here investigated four new [Ru(phen)₂(dppz)]²⁺ derivatives with different bulky quaternary ammonium substituents on the dppz ligand to find relationships between molecular structure and intercalation kinetics, which is considered to be of importance for antitumor applicability. Linear dichroism spectroscopy shows that the enantiomers of the new complexes exhibit very similar binding geometries (intercalation of dppz moiety between adjacent DNA base pairs) as the enantiomers of the parent [Ru(phen)₂(dppz)]²⁺ complex. Absorption spectra and luminescence properties provide further evidence for a final intercalative binding mode which has to be reached by threading of a bulky moiety between the strands of the DNA. Δ-enantiomers of all the new complexes show much slower association and dissociation kinetics than that of a reference complex without a cationic substituent. Kinetics were not very different whether the bulky quaternary group was derived from hexamethylene tetramine or 1,4-diazabicyclo-(2,2,2)octane (DABCO) or whether it had one or two positive charges. However, a complex in which the hexamethylene tetramine substituent is attached via a phenyl group showed a lowered association rate, in addition to an improved quantum yield of luminescence. A second positive charge on the DABCO substituent resulted in a much slower dissociation rate, suggesting that the distance from the Ru-centre and the amount of charge are both important for threading intercalation kinetics.     

Thermodynamic analysis of inducer binding to the lactose repressor protein: contributions of galactosyl hydroxyl groups and beta substituents

Kinetic and equilibrium studies of the binding of modified beta-D-galactoside sugars to the lac repressor were carried out to generate thermodynamic data for protein-inducer interactions. The energetic contributions of the galactosyl hydroxyl groups to binding were assessed by using a series of methyl deoxyfluoro-beta-D-galactosides. The C-3 and C-6 hydroxyls contributed less than or equal to -2.3 and -1.7 +/- 0.3 kcal/mol to the binding free energy change, respectively, whereas the C-4 hydroxyl provided only a nominal contribution (-0.1 +/- 0.2 kcal/mol). Favorable contributions to the total binding free energy change were observed for replacement of O-methyl by S-methyl at the beta-anomeric position and for S-methyl by S-isopropyl. Negative delta H degrees values characteristic of protein-sugar complexes [Quiocho, F. A. (1986) Annu. Rev. Biochem. 55, 287-315] were observed for a series of beta-D-galactosides differing at the beta-glycosidic position. A decrease in delta H degrees of approximately 6 kcal/mol upon replacement of the O-methyl substituent by S-methyl indicates a substantial increase in van der Waals' interactions and/or hydrogen bonding in this region of the ligand binding site. The more favorable free energy change for the binding of the S-isopropyl vs S-methyl compound is due mainly to more positive entropic contributions, consistent with an increase in apolar interactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energetics of sequence-specific protein-DNA association: computational analysis of integrase Tn916 binding to its target DNA

The N-terminal domain of the bacterial integrase Tn916 specifically recognizes the 11 bp DNA target site by positioning the face of a three-stranded beta-sheet into the major groove. Binding is linked to structural adaptation. We have characterized INT-DBD binding to DNA in detail by calorimetry [Milev, S., Gorfe, A., Karshikoff, A., Clubb, R. T., Bosshard, H. R., and Jelesarov, I. (2003) Biochemistry 42, 3481-3491]. Our thermodynamic analysis has indicated that the major driving force of association is the hydrophobic effect while polar interactions contribute less. To gain more comprehensive information about the binding process, we performed a computational analysis of the binding free energy and report here the results. A hybrid molecular mechanics/continuum approach was followed. The total binding free energy is predicted with reasonable accuracy. The calculations confirm that nonpolar effects stabilize the protein-DNA complex while electrostatics opposes binding. Structural changes optimizing surface complementarity are costly in terms of energy. The energetic consequences from the replacement of nine DNA-contacting residues by alanine were investigated. The calculations correctly predict the binding affinity decrease of eight mutations and the destabilizing effect of one wild-type residue. Bulky side chains stabilize the wild-type complex through packing interactions and favorable nonpolar dehydration, but the net nonpolar energy changes do not correlate with the relative affinity loss upon mutation. Discrete protein-DNA electrostatic interactions may be net stabilizing or net destabilizing depending on the local environment. In contrast to nonpolar energy changes, the magnitude of the electrostatic free energy ranks the mutations according to the experimentally measured DeltaDeltaG. Free energy decomposition analysis from a structural perspective leads to detailed information about the thermodynamic strategy used by INT-DBD for sequence-specific DNA binding.     

Absolute binding free energy calculations using molecular dynamics simulations with restraining potentials

The absolute (standard) binding free energy of eight FK506-related ligands to FKBP12 is calculated using free energy perturbation molecular dynamics (FEP/MD) simulations with explicit solvent. A number of features are implemented to improve the accuracy and enhance the convergence of the calculations. First, the absolute binding free energy is decomposed into sequential steps during which the ligand-surrounding interactions as well as various biasing potentials restraining the translation, orientation, and conformation of the ligand are turned "on" and "off." Second, sampling of the ligand conformation is enforced by a restraining potential based on the root mean-square deviation relative to the bound state conformation. The effect of all the restraining potentials is rigorously unbiased, and it is shown explicitly that the final results are independent of all artificial restraints. Third, the repulsive and dispersive free energy contribution arising from the Lennard-Jones interactions of the ligand with its surrounding (protein and solvent) is calculated using the Weeks-Chandler-Andersen separation. This separation also improves convergence of the FEP/MD calculations. Fourth, to decrease the computational cost, only a small number of atoms in the vicinity of the binding site are simulated explicitly, while all the influence of the remaining atoms is incorporated implicitly using the generalized solvent boundary potential (GSBP) method. With GSBP, the size of the simulated FKBP12/ligand systems is significantly reduced, from approximately 25,000 to 2500. The computations are very efficient and the statistical error is small ( approximately 1 kcal/mol). The calculated binding free energies are generally in good agreement with available experimental data and previous calculations (within approximately 2 kcal/mol). The present results indicate that a strategy based on FEP/MD simulations of a reduced GSBP atomic model sampled with conformational, translational, and orientational restraining potentials can be computationally inexpensive and accurate.     

Conformational dynamics and thermodynamics of protein-ligand binding studied by NMR relaxation

Protein conformational dynamics can be critical for ligand binding in two ways that relate to kinetics and thermodynamics respectively. First, conformational transitions between different substates can control access to the binding site (kinetics). Secondly, differences between free and ligand-bound states in their conformational fluctuations contribute to the entropy of ligand binding (thermodynamics). In the present paper, I focus on the second topic, summarizing our recent results on the role of conformational entropy in ligand binding to Gal3C (the carbohydrate-recognition domain of galectin-3). NMR relaxation experiments provide a unique probe of conformational entropy by characterizing bond-vector fluctuations at atomic resolution. By monitoring differences between the free and ligand-bound states in their backbone and side chain order parameters, we have estimated the contributions from conformational entropy to the free energy of binding. Overall, the conformational entropy of Gal3C increases upon ligand binding, thereby contributing favourably to the binding affinity. Comparisons with the results from isothermal titration calorimetry indicate that the conformational entropy is comparable in magnitude to the enthalpy of binding. Furthermore, there are significant differences in the dynamic response to binding of different ligands, despite the fact that the protein structure is virtually identical in the different protein-ligand complexes. Thus both affinity and specificity of ligand binding to Gal3C appear to depend in part on subtle differences in the conformational fluctuations that reflect the complex interplay between structure, dynamics and ligand interactions.     

DNA-platinum interactions in vitro with trans- and cis-Pt(NH3)2Cl2.

In the present study the nature and the hydrolysis of DNA-Pt complexes with the platinum compounds, [Pt(dien)Cl]Cl, trans- and cis-Pt(NH3)2Cl2, using potentiometric chloride determinations, have been investigated. The trans-Pt(NH3)2Cl2 and the [Pt(dien)Cl]Cl react with the GC planes at the N7(G) sites, while the cis-Pt(NH3)2Cl2 compound reacts with the GC planes and forms a chelate by using the N7(G) and O6(G) sites. The complex is a specific 1:1 Pt:DNA adduct. The platinum atom in cis-Pt(NH3)2Cl2 liberates both chlorine atoms on chelation. A mechanism for the in vivo antitumor activity of the cis-Pt(NH3)2Cl2 is proposed and the structure activity relationship is discussed.     

Theoretical studies of cis-Pt(II)-diammine binding to duplex DNA

The binding of cis-Pt(II) diammine (cis-DP) to double-stranded DNA was studied with several kinked conformations that can accommodate the formation of a square planar complex. Molecular mechanics (MM) calculations were performed to optimize the molecular fit. These results were combined with quantum mechanical (QM) calculations to ascertain the relative energetics of ligand binding through water vs direct binding of the phosphate to the ammine and platinum, and to guide the selection of DNA conformations to model complex formation. Based on QM and MM calculations, models are proposed that may be characterized by several general features. A structure involving hydrogen bonding between each ammine and distinct adjacent phosphate groups, referred to as closed conformation (CC), has already been reported. This is also found in the crystal structure of small dimers. We report alternative conformations that may be important in platination of duplex DNA. They are characterized by an intermediate conformation (IC), involving hydrogen bonding between one ammine and phosphate group, and an open conformation (OC), without ammine phosphate hydrogen bonding. The IC and OC can be stabilized by water bridges in the space between the ammine and the phosphate groups. Sugar puckers alternate from the type C(2')-endo or C(1')-exo (S), to the type C(3')-endo or C(2')-exo (N), with intermediate types near O(1')-endo (O). In general, the sugar puckers alternate from S to N to S through the platinated region (3'-TpG*pG*p-5'), with the complexed strand exhibiting, (3')-S*-N*-S-(5') alternation, while the complementary strand shows either (3')-S*-N*-S-(5') or (3')-S*-N*-O-(5') alternation. In both the OC and IC, a hydrogen bond is found between the ammine and O4(T) on thymine (T) at the (3') end, adjacent to the complex site. There is a continuous range of backbone conformations through the platinated region which relate the OC to the IC. The models presented suggest that the dynamics of the binding of the cis-Pt(II)-diammines to adjacent N7(G) in double-stranded DNA may encompass several conformational possibilities, and that water bridges may play a roll in supporting open and intermediate conformations. Proton-proton distances are reported to assist in the experimental determination of conformations.     

The B-ring substituent at C-7 of colchicine and the alpha-C-terminus of tubulin communicate through the "tail-body" interaction

The carboxy terminals of alphabeta-tubulins are flexible regions rich in acidic amino acid residues that play an inhibitory role in the polymerization of tubulin to microtubules. We have shown that the binding of colchicine and its B-ring analogs (with C-7 substituents) to tubulin are pH sensitive and have high activation energies. Under identical conditions, the binding of analogs without C-7 substituents is pH independent and has lower activation energy. Beta-C-terminus-truncated tubulin (alphabeta(s)) shows similar pH sensitivity and activation energy to native tubulin (alphabeta). Removal of the C-termini of both subunits of tubulin (alpha(s)beta(s)) or the binding of a basic peptide P2 to the negatively charged alpha-C-terminus of tubulin causes a colchicine-tubulin interaction independent of pH with a low activation energy. Tubulin dimer structure shows that the C-terminal alpha-tail is too far from the colchicine binding site to interact directly with the bound colchicine. Therefore, it is likely that the interaction of the alpha-C-terminus with the main body of tubulin indirectly affects the colchicine-tubulin interaction via conformational changes in the main body. We therefore conclude that in the presence of tail-body interaction, a B-ring substituent makes contact with the alpha-tubulin and induces significant conformational changes in alpha-tubulin.