Favorable Binding Free Energy Research Articles

Molecular dynamics and free energy analysis of neuraminidase-ligand interactions.

We report molecular dynamics calculations of neuraminidase in complex with an inhibitor, 4-amino-2-deoxy-2,3-didehydro-N-acetylneuraminic acid (N-DANA), with subsequent free energy analysis of binding by using a combined molecular mechanics/continuum solvent model approach. A dynamical model of the complex containing an ionized Glu119 amino acid residue is found to be consistent with experimental data. Computational analysis indicates a major van der Waals component to the inhibitor-neuraminidase binding free energy. Based on the N-DANA/neuraminidase molecular dynamics trajectory, a perturbation methodology was used to predict the binding affinity of related neuraminidase inhibitors by using a force field/Poisson-Boltzmann potential. This approach, incorporating conformational search/local minimization schemes with distance-dependent dielectric or generalized Born solvent models, correctly identifies the most potent neuraminidase inhibitor. Mutation of the key ligand four-substituent to a hydrogen atom indicates no favorable binding free energy contribution of a hydroxyl group; conversely, cationic substituents form favorable electrostatic interactions with neuraminidase. Prospects for further development of the method as an analysis and rational design tool are discussed.

Identification of Substrate Binding Sites in Enzymes by Computational Solvent Mapping

Enzyme structures determined in organic solvents show that most organic molecules cluster in the active site, delineating the binding pocket. We have developed algorithms to perform solvent mapping computationally, rather than experimentally, by placing molecular probes (small molecules or functional groups) on a protein surface, and finding the regions with the most favorable binding free energy. The method then finds the consensus site that binds the highest number of different probes. The probe–protein interactions at this site are compared to the intermolecular interactions seen in the known complexes of the enzyme with various ligands (substrate analogs, products, and inhibitors). We have mapped thermolysin, for which experimental mapping results are also available, and six further enzymes that have no experimental mapping data, but whose binding sites are well characterized. With the exception of haloalkane dehalogenase, which binds very small substrates in a narrow channel, the consensus site found by the mapping is always a major subsite of the substrate-binding site. Furthermore, the probes at this location form hydrogen bonds and non-bonded interactions with the same residues that interact with the specific ligands of the enzyme. Thus, once the structure of an enzyme is known, computational solvent mapping can provide detailed and reliable information on its substrate-binding site. Calculations on ligand-bound and apo structures of enzymes show that the mapping results are not very sensitive to moderate variations in the protein coordinates.

Increased Backbone Mobility in β-Barrel Enhances Entropy Gain Driving Binding of N-TIMP-1 to MMP-3

The high-affinity inhibition of stromelysin 1 (MMP-3) by tissue inhibitor of metalloproteinases 1 (TIMP-1) helps control tissue remodeling and tumor development. The interaction of N-TIMP-1 with the catalytic domain of MMP-3 has been investigated by titration calorimetry and 15N NMR. Their unfavorable enthalpy of binding of +6.5kcalmol−1 is unusual among protein–protein associations, deviates from structure-based prediction, and is compensated by a net entropy increase providing at least 18kcalmol−1 of favorable free energy of binding at a 1M reference state. The small heat capacity of binding agrees well with the heat capacity predicted from 65% of the surface buried on binding being polar, and suggests that the hydrophobic effect can account for only part of the entropy of binding. Using NMR, binding-induced changes in the backbone of N-TIMP-1 were checked as one possible source of conformational entropy changes. MMP binding slightly increases rigidity in some contact sites in TIMP-1 but increases mobility remotely in the otherwise rigid β-barrel core of N-TIMP-1, increasing 15N relaxation evidence of pico- to nanosecond and micro- to millisecond fluctuations of β-strands A–F. Residual dipolar couplings suggest dynamic deviations from X-ray coordinates of the complex. These suggest that the β-barrel has small backbone conformational fluctuations, while segments of strands βB, βE and βF might experience fluctuations only in their backbone environment. This is a distinctive example of affinity between two well-structured proteins being enhanced by increased conformational entropy in the reservoir of a folding core.

Charge optimization leads to favorable electrostatic binding free energy.

Variational optimization of molecular electrostatic charge distributions is a tool for the study of association reactions of molecules in solution. In principle, this method can be used in drug design and protein folding to analyze and improve molecular interactions and to provide electrostatic templates for molecular design. This optimization problem reduces to an inverse source problem in classical electrostatics, where the sources are determined by a combination of external and self-polarization potentials. In this paper, we show that the electrostatic portion of the free energy of association for electrostatically optimized molecules has an upper bound of zero in many situations of physical interest. That is, variational optimization provides a ligand-charge distribution that contributes favorably to the energetics of binding, even in a strongly polar medium. This stabilizing effect on association reactions is contrary to the usual role of electrostatics in aqueous complexes, in which desolvation effects generally dominate. We also show the existence and nonuniqueness of the variational solution and make a connection to the electrostatic image charge problem.

Optimizing electrostatic affinity in ligand–receptor binding: Theory, computation, and ligand properties

The design of a tight-binding molecular ligand involves a tradeoff between an unfavorable electrostatic desolvation penalty incurred when the ligand binds a receptor in aqueous solution and the generally favorable intermolecular interactions made in the bound state. Using continuum electrostatic models we have developed a theoretical framework for analyzing this problem and have shown that the ligand-charge distribution can be optimized to produce the most favorable balance of these opposing free energy contributions [L.-P. Lee and B. Tidor, J. Chem. Phys. 106, 8681 (1997)]. Herein the theoretical framework is extended and calculations are performed for a wide range of model receptors. We examine methods for computing optimal ligands (including cases where there is conformational change) and the resulting properties of optimized ligands. In particular, indicators are developed to aid in the determination of the deficiencies in a specific ligand or basis. A connection is established between the optimization problem here and a generalized image problem, from which an inverse-image basis set can be defined; this basis is shown to perform very well in optimization calculations. Furthermore, the optimized ligands are shown to have favorable electrostatic binding free energies (in contrast to many natural ligands), there is a strong correlation between the receptor desolvation penalty and the optimized binding free energy for fixed geometry, and the ligand and receptor cannot generally be mutually optimal. Additionally, we introduce the display of complementary desolvation and interaction potentials and the deviation of their relationship from ideal as a useful tool for judging effective complementarity. Scripts for computing and displaying these potentials with GRASP are available at http://mit.edu/tidor.

Equilibrium and kinetic parameters of the sequence-specific interaction of Escherichia coli RNA polymerase with nontemplate strand oligodeoxyribonucleotides.

The specific recognition by Escherichia coli RNA polymerase of single-stranded oligodeoxyribonucleotides (oligos) with the sequence of the -10 promoter region on the nontemplate strand has been studied. Binding was monitored by observing the increase in fluorescence of 2-aminopurine residues incorporated in the oligos. The effects of salt on the rates of formation and dissociation of RNA polymerase.oligo complexes are relatively small, from which we conclude that electrostatic interactions contribute minimally to the favorable binding free energy. From the convex temperature dependence of ln Ka (Ka is the equilibrium association constant), we infer that a large apparent negative heat capacity, of 1-2 kcal M-1 K-1, accompanies complex formation, which is interpreted as due to a conformational change in RNA polymerase. Contrary to expectation, the forward rate constant for binding of oligos is more than 10-fold smaller than that for open complex formation at strong promoters. This suggests that in comparison to an oligo, promoter DNA may be better able to accelerate this required conformational change in the RNA polymerase. Oligo binding was shown to compete with the interaction between RNA polymerase and promoters, indicating that the two bind to overlapping sites on the RNA polymerase

Ultratight DNA binding of a new bisintercalating anthracycline antibiotic.

Differential scanning calorimetry and absorption spectroscopy were used to characterize the interaction of the new bisintercalating anthracycline antibiotic, WP631, with DNA. The method of continuous variations revealed five distinct binding modes for WP631, corresponding to 6, 3, 1.3, 0.5, and 0.25 mol of base pairs (bp) per mole of ligand. The binding of one drug to 6 bp corresponds to the bisintercalative binding mode determined previously, and was the mode studied in detail. UV melting experiments and differential scanning calorimetry were used to measure the ultratight binding of WP631 to DNA. The binding constant for the interaction of WP631 with herring sperm DNA was determined to be 3.1 (+/- 0.2) x 10(11) M-1 at 20 degrees C. The large, favorable binding free energy of -15.3 kcal mol-1 was found to result from a large, negative enthalpic contribution of -30.2 kcal mol-1. DNA melting curves at different concentrations of WP631 were fitted to McGhee's model of DNA melting in the presence of ligands, yielding an independent estimate of DNA binding parameters. The salt dependence of the WP631 binding constant was examined, yielding a slope SK = delta (log K)/delta (log[Na+]) = 1.63. The observed salt dependence of the equilibrium constant, interpreted according to polyelectrolyte theory, indicates that there is a significant nonpolyelectrolyte contribution to the binding free energy. DNA melting studies using a homogeneous 214 bp DNA fragment showed that WP631 binds preferentially to the GC-rich region of the DNA.

Structure-based design of a new bisintercalating anthracycline antibiotic.

A new bisintercalating anthracycline antibiotic, WP631, has been designed and synthesized. The rational design of the new compound was based upon the geometry of monomeric anthracyclines bound to DNA oligonucleotides observed in high-resolution crystal structures. Monomeric units of daunorubicin have been linked through their reactive 3' NH2 substituents on the daunosamine moieties to form the new bisanthracycline WP631. Viscosity studies confirmed that WP631 binds to DNA by bisintercalation. Differential scanning calorimetry and UV melting experiments were used to measure the ultratight binding of WP631 to DNA. The binding constant for the interaction of WP631 with herring sperm DNA was determined to be 2.7 x 10(11) M-1 at 20 degrees C. The large, favorable binding free energy of -15.3 kcal mol-1 was found to result from a large, negative enthalpic contribution of -30.2 kcal mol-1. A molecular model was generated that shows the favorable stereochemical fit of the linker in the DNA minor groove. The cytotoxicity of WP631 was compared to that of doxorubicin using MCF-7-sensitive and MCF-7/VP-16 MRP-mediated multidrug-resistant cell lines. These initial studies showed that while WP631 is slightly less cytotoxic than doxorubicin in the sensitive cell line, it appears to overcome MRP-mediated multidrug resistance and was much more cytotoxic against the MCF-7/VP-16 cell line than was doxorubicin. The design of new potential anticancer agents based on known structural principles was found to produce a compound with significantly increased DNA binding affinity and with interesting biological activity.

Computational combinatorial ligand design: application to human alpha-thrombin.

A new method is presented for computer-aided ligand design by combinatorial selection of fragments that bind favorably to a macromolecular target of known three-dimensional structure. Firstly, the multiple-copy simultaneous-search procedure (MCSS) is used to exhaustively search for optimal positions and orientations of functional groups on the surface of the macromolecule (enzyme or receptor fragment). The MCSS minima are then sorted according to an approximated binding free energy, whose solvation component is expressed as a sum of separate electrostatic and nonpolar contributions. The electrostatic solvation energy is calculated by the numerical solution of the linearized Poisson-Boltzmann equation, while the nonpolar contribution to the binding free energy is assumed to be proportional to the loss in solvent-accessible surface area. The program developed for computational combinatorial ligand design (CCLD) allows the fast and automatic generation of a multitude of highly diverse compounds, by connecting in a combinatorial fashion the functional groups in their minimized positions. The fragments are linked as two atoms may be either fused, or connected by a covalent bond or a small linker unit. To avoid the combinatorial explosion problem, pruning of the growing ligand is performed according to the average value of the approximated binding free energy of its fragments. The method is illustrated here by constructing candidate ligands for the active site of human alpha-thrombin. The MCSS minima with favorable binding free energy reproduce the interaction patterns of known inhibitors. Starting from these fragments, CCLD generates a set of compounds that are closely related to high-affinity thrombin inhibitors. In addition, putative ligands with novel binding motifs are suggested. Probable implications of the MCSS-CCLD approach for the evolving scenario of drug discovery are discussed.

Binding energy and enzymatic catalysis

The authors discuss the fundamental role that the favorable free energy of binding of the rate-determining transition state plays in catalysis and review the principle that many catalytic factors a...

Thermodynamics of the daunomycin-DNA interaction: ionic strength dependence of the enthalpy and entropy.

AbstractFluorescence and absorbance methods were used to study the interaction of daunomycin with calf‐thymus DNA over a wide range of temperatures and NaCl concentrations. van't Hoff analysis provided estimates for the enthalpy of the binding reaction over the NaCl range of 0.05–1.0 M. Daunomycin binding is exothermic over this entire range, and the favorable binding free energy arises primarily from the large, negative enthalpy. Both the enthalpy change and entropy change are strong functions of ionic strength. Possible molecular contributions to the enthalpy and entropy are discussed, leading to the tentative conclusion that hydrogen‐bonding interactions at the interacalation site are the primary contributors to the observed thermodynamic parameters. The dependence of the enthalpy on the ionic strength is well beyond the predictions of current polyelectrolyte theory and cannot be fully accounted for. The enthalpy and entropy changes observed compensate one another to produce relatively small free‐energy changes over the range of solution conditions studied.

Ion effects on the lac repressor--operator equilibrium.

The effects of ions on the interaction of lac repressor protein and operator DNA have been studied by the membrane filter technique. The equilibrium association constant was determined as a function of monovalent and divalent cation concentrations, anions, and pH. The binding of repressor and operator is extremely sensitive to the ionic environment. The dependence of the observed equilibrium constant on salt concentration is analyzed according to the binding theory of Record et al. [Record, M. T., Jr., Lohman, T. M., & deHaseth, P. L. (1976) J. Mol. Biol. 107, 145]. The number of ionic interactions in repressor--operator complex is deduced from the slopes of the linear log-log plots. About 11 ionic interactions are formed between repressor and DNA phosphates at pH 7.4 and about 9 ionic interactions at pH 8.0, in reasonable agreement with previous estimates. A favorable nonelectrostatic binding free energy of about 9-12 kcal/mol is estimated from the extrapolated equilibrium constants at the 1 M standard state. The values are in good accord with recent results for the salt-independent binding of repressor core and operator DNA. The effects of pH on the repressor--operator interaction are small, and probably result from titration of functional groups in the DNA-binding site of the protein. For monovalent salts, the equilibrium constant is slightly dependent on cation type and highly dependent on anion type. At constant salt concentration, the equilibrium constant decreases about 10000-fold in the order CH3CO2- greater than or equal to F- greater than Cl- greater than Br- greater than NO3- greater than SCN- greater than I-. The wide range of accessible equilibrium constants provides a useful tool for in vitro studies of the repressor--operator interaction.