Impact of ligand and protein desolvation on ligand binding to the S1 pocket of thrombin

In the present study, we investigate the impact of a tightly bound water molecule on ligand binding in the S1 pocket of thrombin. The S1 pocket contains a deeply buried deprotonated aspartate residue (Asp189) that is, due to its charged state, well hydrated in the uncomplexed state. We systematically studied the importance of this water molecule by evaluating a series of ligands that contains pyridine-type P1 side chains that could potentially alter the binding properties of this water molecule. All of the pyridine derivatives retain the original hydration state albeit sometimes with a slight perturbance. In order to prevent a direct H-bond formation with Asp189, and to create a permanent positive charge on the P1 side chain that is positioned adjacent to the Asp189 carboxylate anion, we methylated the pyridine nitrogen. This methylation resulted in displacement of water but was accompanied by a loss in binding affinity. Quantum chemical calculations of the ligand solvation free energy showed that the positively charged methylpyridinium derivatives suffer a large penalty of desolvation upon binding. Consequently, they have a substantially less favorable enthalpy of binding. In addition to the ligand desolvation penalty, the hydration shell around Asp189 has to be overcome, which is achieved in nearly all pyridinium derivatives. Only for the ortho derivative is a partial population of a water next to Asp189 found. Possibly, the gain of electrostatic interactions between the charged P1 side chain and Asp189 helps to compensate for the desolvation penalty. In all uncharged pyridine derivatives, the solvation shell remains next to Asp189, partly mediating interactions between ligand and protein. In the case of the para-pyridine derivative, a strongly disordered cluster of water sites is observed between ligand and Asp189.     

Optimization of binding electrostatics: charge complementarity in the barnase-barstar protein complex

Theoretical and experimental studies have shown that the large desolvation penalty required for polar and charged groups frequently precludes their involvement in electrostatic interactions that contribute strongly to net stability in the folding or binding of proteins in aqueous solution near room temperature. We have previously developed a theoretical framework for computing optimized electrostatic interactions and illustrated use of the algorithm with simplified geometries. Given a receptor and model assumptions, the method computes the ligand-charge distribution that provides the most favorable balance of desolvation and interaction effects on binding. In this paper the method has been extended to treat complexes using actual molecular shapes. The barnase-barstar protein complex was investigated with barnase treated as a target receptor. The atomic point charges of barstar were varied to optimize the electrostatic binding free energy. Barnase and natural barstar form a tight complex (K(d) approximately 10(-14) M) with many charged and polar groups near the interface that make this a particularly relevant system for investigating the role of electrostatic effects on binding. The results show that sets of barstar charges (resulting from optimization with different constraints) can be found that give rise to relatively large predicted improvements in electrostatic binding free energy. Principles for enhancing the effect of electrostatic interactions in molecular binding in aqueous environments are discussed in light of the optima. Our findings suggest that, in general, the enhancements in electrostatic binding free energy resulting from modification of polar and charged groups can be substantial. Moreover, a recently proposed definition of electrostatic complementarity is shown to be a useful tool for examining binding interfaces. Finally, calculational results suggest that wild-type barstar is closer to being affinity optimized than is barnase for their mutual binding, consistent with the known roles of these proteins.     

Structural determinants of trypsin affinity and specificity for cationic inhibitors

The binding free energies of four inhibitors to bovine beta-trypsin are calculated. The inhibitors use either ornithine, lysine, or arginine to bind to the S1 specificity site. The electrostatic contribution to binding free energy is calculated by solving the finite difference Poisson-Boltzmann equation, the contribution of nonpolar interactions is calculated using a free energy-surface area relationship and the loss of conformational entropy is estimated both for trypsin and ligand side chains. Binding free energy values are of a reasonable magnitude and the relative affinity of the four inhibitors for trypsin is correctly predicted. Electrostatic interactions are found to oppose binding in all cases. However, in the case of ornithine- and lysine-based inhibitors, the salt bridge formed between their charged group and the partially buried carboxylate of Asp189 is found to stabilize the complex. Our analysis reveals how the molecular architecture of the trypsin binding site results in highly specific recognition of substrates and inhibitors. Specifically, partially burying Asp189 in the inhibitor-free enzyme decreases the penalty for desolvation of this group upon complexation. Water molecules trapped in the binding interface further stabilize the buried ion pair, resulting in a favorable electrostatic contribution of the ion pair formed with ornithine and lysine side chains. Moreover, all side chains that form the trypsin specificity site are partially buried, and hence, relatively immobile in the inhibitor-free state, thus reducing the entropic cost of complexation. The implications of the results for the general problem of recognition and binding are considered. A novel finding in this regard is that like charged molecules can have electrostatic contributions to binding that are more favorable than oppositely charged molecules due to enhanced interactions with the solvent in the highly charged complex that is formed.     

Prediction of Protein-Protein Interaction Sites Using Electrostatic Desolvation Profiles

Protein-protein complex formation involves removal of water from the interface region. Surface regions with a small free energy penalty for water removal or desolvation may correspond to preferred interaction sites. A method to calculate the electrostatic free energy of placing a neutral low-dielectric probe at various protein surface positions has been designed and applied to characterize putative interaction sites. Based on solutions of the finite-difference Poisson equation, this method also includes long-range electrostatic contributions and the protein solvent boundary shape in contrast to accessible-surface-area-based solvation energies. Calculations on a large set of proteins indicate that in many cases (>90%), the known binding site overlaps with one of the six regions of lowest electrostatic desolvation penalty (overlap with the lowest desolvation region for 48% of proteins). Since the onset of electrostatic desolvation occurs even before direct protein-protein contact formation, it may help guide proteins toward the binding region in the final stage of complex formation. It is interesting that the probe desolvation properties associated with residue types were found to depend to some degree on whether the residue was outside of or part of a binding site. The probe desolvation penalty was on average smaller if the residue was part of a binding site compared to other surface locations. Applications to several antigen-antibody complexes demonstrated that the approach might be useful not only to predict protein interaction sites in general but to map potential antigenic epitopes on protein surfaces.     

Free energy landscapes of encounter complexes in protein-protein association

We report the computer generation of a high-density map of the thermodynamic properties of the diffusion-accessible encounter conformations of four receptor-ligand protein pairs, and use it to study the electrostatic and desolvation components of the free energy of association. Encounter complex conformations are generated by sampling the translational/rotational space of the ligand around the receptor, both at 5-A and zero surface-to-surface separations. We find that partial desolvation is always an important effect, and it becomes dominant for complexes in which one of the reactants is neutral or weakly charged. The interaction provides a slowly varying attractive force over a small but significant region of the molecular surface. In complexes with no strong charge complementarity this region surrounds the binding site, and the orientation of the ligand in the encounter conformation with the lowest desolvation free energy is similar to the one observed in the fully formed complex. Complexes with strong opposite charges exhibit two types of behavior. In the first group, represented by barnase/barstar, electrostatics exerts strong orientational steering toward the binding site, and desolvation provides some added adhesion within the local region of low electrostatic energy. In the second group, represented by the complex of kallikrein and pancreatic trypsin inhibitor, the overall stability results from the rather nonspecific electrostatic attraction, whereas the affinity toward the binding region is determined by desolvation interactions.     

Probing molecular docking in a charged model binding site

A model binding site was used to investigate charge-charge interactions in molecular docking. This simple site, a small (180A(3)) engineered cavity in cyctochrome c peroxidase (CCP), is negatively charged and completely buried from solvent, allowing us to explore the balance between electrostatic energy and ligand desolvation energy in a system where many of the common approximations in docking do not apply. A database with about 5300 molecules was docked into this cavity. Retrospective testing with known ligands and decoys showed that overall the balance between electrostatic interaction and desolvation energy was captured. More interesting were prospective docking scre"ens that looked for novel ligands, especially those that might reveal problems with the docking and energy methods. Based on screens of the 5300 compound database, both high-scoring and low-scoring molecules were acquired and tested for binding. Out of 16 new, high-scoring compounds tested, 15 were observed to bind. All of these were small heterocyclic cations. Binding constants were measured for a few of these, they ranged between 20microM and 60microM. Crystal structures were determined for ten of these ligands in complex with the protein. The observed ligand geometry corresponded closely to that predicted by docking. Several low-scoring alkyl amino cations were also tested and found to bind. The low docking score of these molecules owed to the relatively high charge density of the charged amino group and the corresponding high desolvation penalty. When the complex structures of those ligands were determined, a bound water molecule was observed interacting with the amino group and a backbone carbonyl group of the cavity. This water molecule mitigates the desolvation penalty and improves the interaction energy relative to that of the "naked" site used in the docking screen. Finally, six low-scoring neutral molecules were also tested, with a view to looking for false negative predictions. Whereas most of these did not bind, two did (phenol and 3-fluorocatechol). Crystal structures for these two ligands in complex with the cavity site suggest reasons for their binding. That these neutral molecules do, in fact bind, contradicts previous results in this site and, along with the alkyl amines, provides instructive false negatives that help identify weaknesses in our scoring functions. Several improvements of these are considered.     

Conformational selection through electrostatics: Free energy simulations of GTP and GDP binding to archaeal initiation factor 2

Archaeal Initiation Factor 2 is a GTPase involved in protein biosynthesis. In its GTP-bound, "ON" conformation, it binds an initiator tRNA and carries it to the ribosome. In its GDP-bound, "OFF" conformation, it dissociates from tRNA. To understand the specific binding of GTP and GDP and their dependence on the conformational state, molecular dynamics free energy simulations were performed. The ON state specificity was predicted to be weak, with a GTP/GDP binding free energy difference of -1 kcal/mol, favoring GTP. The OFF state specificity is larger, 4 kcal/mol, favoring GDP. The overall effects result from a competition among many interactions in several complexes. To interpret them, we use a simpler, dielectric continuum model. Several effects are robust with respect to the model details. Both nucleotides have a net negative charge, so that removing them from solvent into the binding pocket carries a desolvation penalty, which is large for the ON state, and strongly disfavors GTP binding compared to GDP. Short-range interactions between the additional GTP phosphate group and ionized sidechains in the binding pocket offset most, but not all of the desolvation penalty; more distant groups also contribute significantly, and the switch 1 loop only slightly. The desolvation penalty is lower for the more open, wetter OFF state, and the GTP/GDP difference much smaller. Short-range interactions in the binding pocket and with more distant groups again make a significant contribution. Overall, the simulations help explain how conformational selection is achieved with a single phosphate group.     

Calculation of binding free energies for non-zinc chelating pyrimidine dicarboxamide inhibitors with MMP-13

All-atom molecular dynamics (MD) simulations in both explicit and implicit solvent, followed by MM-GBSA energy analysis, have been used to estimate binding free energies of four pyrimidine dicarboxamide inhibitors with human collagenase-3 (MMP-13) for comparison with experimental activities. Energetic analysis reveals that affinity is driven primarily by favorable van der Waals interactions and burial of total surface area. The computed effects of desolvation, as a function of ligand structure, quantitatively show that hydrophilic derivatives pay greater penalties upon binding than their related more hydrophobic analogs.     

Thermodynamic penalty arising from burial of a ligand polar group within a hydrophobic pocket of a protein receptor

Here, we examine the thermodynamic penalty arising from burial of a polar group in a hydrophobic pocket that forms part of the binding-site of the major urinary protein (MUP-I). X-ray crystal structures of the complexes of octanol, nonanol and 1,8 octan-diol indicate that these ligands bind with similar orientations in the binding pocket. Each complex is characterised by a bridging water molecule between the hydroxyl group of Tyr120 and the hydroxyl group of each ligand. The additional hydroxyl group of 1,8 octan-diol is thereby forced to reside in a hydrophobic pocket, and isothermal titration calorimetry experiments indicate that this is accompanied by a standard free energy penalty of +21 kJ/mol with respect to octanol and +18 kJ/mol with respect to nonanol. Consideration of the solvation thermodynamics of each ligand enables the "intrinsic" (solute-solute) interaction energy to be determined, which indicates a favourable enthalpic component and an entropic component that is small or zero. These data indicate that the thermodynamic penalty to binding derived from the unfavourable desolvation of 1,8 octan-diol is partially offset by a favourable intrinsic contribution. Quantum chemical calculations suggest that this latter contribution derives from favourable solute-solute dispersion interactions.     

Algorithms for computational solvent mapping of proteins

Computational mapping methods place molecular probes (small molecules or functional groups) on a protein surface to identify the most favorable binding positions by calculating an interaction potential. We have developed a novel computational mapping program called CS-Map (computational solvent mapping of proteins), which differs from earlier mapping methods in three respects: (i) it initially moves the ligands on the protein surface toward regions with favorable electrostatics and desolvation, (ii) the final scoring potential accounts for desolvation, and (iii) the docked ligand positions are clustered, and the clusters are ranked on the basis of their average free energies. To understand the relative importance of these factors, we developed alternative algorithms that use the DOCK and GRAMM programs for the initial search. Because of the availability of experimental solvent mapping data, lysozyme and thermolysin are considered as test proteins. Both DOCK and GRAMM speed up the initial search, and the combined algorithms yield acceptable mapping results. However, the DOCK-based approaches place the consensus site farther from its experimentally determined position than CS-Map, primarily because of the lack of a solvation term in the initial search. The GRAMM-based program also finds the correct consensus site for thermolysin. We conclude that good sampling is the most important requirement for successful mapping, but accounting for desolvation and clustering of ligand positions also help to reduce the number of false positives.     

Thermodynamic analysis of binding between mouse major urinary protein-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole

The mouse pheromone 2-sec-butyl-4,5-dihydrothiazole (SBT) binds to an occluded, nonpolar cavity in the mouse major urinary protein-I (MUP-I). The thermodynamics of this interaction have been characterized using isothermal titration calorimetry (ITC). MUP-I-SBT binding is accompanied by a large favorable enthalpy change (DeltaH = -11.2 kcal/mol at 25 degrees C), an unfavorable entropy change (-TDeltaS = 2.8 kcal/mol at 25 degrees C), and a negative heat capacity change [DeltaC(p)() = -165 cal/(mol K)]. Thermodynamic analysis of binding between MUP-I and several 2-alkyl-4,5-dihydrothiazole ligands indicated that the alkyl chain contributes more favorably to the enthalpy and less favorably to the entropy of binding than would be expected on the basis of the hydrophobic desolvation of short-chain alcohols. However, solvent transfer experiments indicated that desolvation of SBT is accompanied by a net unfavorable change in enthalpy (DeltaH = +1.0 kcal/mol) and favorable change in entropy (-TDeltaS = -1.8 kcal/mol). These results are discussed in terms of the possible physical origins of the binding thermodynamics, including (1) hydrophobic desolvation of both the protein and the ligand, (2) formation of a buried water-mediated hydrogen bond network between the protein and ligand, (3) formation of strong van der Waals interactions, and (4) changes in the structure, dynamics, and/or hydration of the protein upon binding.     

Synchronized conformational fluctuations and binding site desolvation during molecular recognition

Binding site desolvation is a poorly understood prerequisite to ligand binding. Although structural fluctuations may be expected to have an important role, little is known about which fluctuations are important or the mechanism by which they promote desolvation. This investigation examines whether and how specific structural fluctuations contribute to desolvation of the ligand binding site in glycopeptide antibiotics. Backbone peptide group rotations in vancomycin, known to occur by experimental observation, were examined in this work with a two-dimensional adaptive umbrella sampling molecular dynamics simulation technique. Results indicate that energetic barriers to rotation are relatively small for two of the peptide groups intimately involved in ligand recognition. When they occur, these rotations strip water molecules away from key hydrogen bond donors and simultaneously cause significant distortions in the macrocyclic rings of the antibiotic that force water into and out of the binding site. Both events are intricately synchronized on the molecular level and have consequences that are clearly necessary to prepare the binding site for receiving a ligand. These results suggest that previously reported observations concerning structural dynamics and binding kinetics in these compounds are mechanistically linked, and they illustrate a heretofore unrecognized degree of preorganization, complexity, and synchronization that may be involved in specific molecular recognition. They also suggest that strategies for increasing antibiotic affinity through covalent dimerization may be counterproductive.     

Continuum electrostatic model for the binding of cytochrome c2 to the photosynthetic reaction center from Rhodobacter sphaeroides

Electrostatic interactions are important for protein-protein association. In this study, we examined the electrostatic interactions between two proteins, cytochrome c(2) (cyt c(2)) and the reaction center (RC) from the photosynthetic bacterium Rhodobacter sphaeroides, that function in intermolecular electron transfer in photosynthesis. Electrostatic contributions to the binding energy for the cyt c(2)-RC complex were calculated using continuum electrostatic methods based on the recent cocrystal structure [Axelrod, H. L., et al. (2002) J. Mol. Biol. 319, 501-515]. Calculated changes in binding energy due to mutations of charged interface residues agreed with experimental results for a protein dielectric constant epsilon(in) of 10. However, the electrostatic contribution to the binding energy for the complex was close to zero due to unfavorable desolvation energies that compensate for the favorable Coulomb attraction. The electrostatic energy calculated as a function of displacement of the cyt c(2) from the bound position showed a shallow minimum at a position near but displaced from the cocrystal configuration. These results show that although electrostatic steering is present, other short-range interactions must be present to contribute to the binding energy and to determine the structure of the complex. Calculations made to model the experimental data on association rates indicate a solvent-separated transition state for binding in which the cyt c(2) is displaced approximately 8 A above its position in the bound complex. These results are consistent with a two-step model for protein association: electrostatic docking of the cyt c(2) followed by desolvation to form short-range van der Waals contacts for rapid electron transfer.     

DAPI binding to the DNA minor groove: a continuum solvent analysis

A continuum solvent model based on the generalized Born (GB) or finite-difference Poisson-Boltzmann (FDPB) approaches has been employed to compare the binding of 4'-6-diamidine-2-phenyl indole (DAPI) to the minor groove of various DNA sequences. Qualitative agreement between the results of GB and FDPB approaches as well as between calculated and experimentally observed trends regarding the sequence specificity of DAPI binding to B-DNA was obtained. Calculated binding energies were decomposed into various contributions to solvation and DNA-ligand interaction. DNA conformational adaptation was found to make a favorable contribution to the calculated total interaction energy but did not change the DAPI binding affinity ranking of different DNA sequences. The calculations indicate that closed complex formation is mainly driven by nonpolar contributions and was found to be disfavored electrostatically due to a desolvation penalty that outbalances the attractive Coulomb interaction. The calculated penalty was larger for DAPI binding to GC-rich sequences compared with AT-rich target sequences and generally larger for the FDPB vs the GB continuum model. A radial interaction profile for DAPI at different distances from the DNA minor groove revealed an electrostatic energy minimum a few Angstroms farther away from the closed binding geometry. The calculated electrostatic interaction up to this distance is attractive and it may stabilize a nonspecific binding arrangement.     

Merging the binding sites of aldose and aldehyde reductase for detection of inhibitor selectivity-determining features

Inhibition of human aldose reductase (ALR2) evolved as a promising therapeutic concept to prevent late complications of diabetes. As well as appropriate affinity and bioavailability, putative inhibitors should possess a high level of selectivity for ALR2 over the related aldehyde reductase (ALR1). We investigated the selectivity-determining features by gradually mapping the residues deviating between the binding pockets of ALR1 and ALR2 into the ALR2 binding pocket. The resulting mutational constructs of ALR2 (eight point mutations and one double mutant) were probed for their influence towards ligand selectivity by X-ray structure analysis of the corresponding complexes and isothermal titration calorimetry (ITC). The binding properties of these mutants were evaluated using a ligand set of zopolrestat, a related uracil derivative, IDD388, IDD393, sorbinil, fidarestat and tolrestat. Our study revealed induced-fit adaptations within the mutated binding site as an essential prerequisite for ligand accommodation related to the selectivity discrimination of the ligands. However, our study also highlights the limits of the present understanding of protein-ligand interactions. Interestingly, binding site mutations not involved in any direct interaction to the ligands in various cases show significant effects towards their binding thermodynamics. Furthermore, our results suggest the binding site residues deviating between ALR1 and ALR2 influence ligand affinity in a complex interplay, presumably involving changes of dynamic properties and differences of the solvation/desolvation balance upon ligand binding.     

Cooperative binding of DNA and CBFbeta to the Runt domain of the CBFalpha studied via MD simulations

The Runt domain (RD) is the DNA-binding region of the Runx genes. A related protein, known as core binding factor β (CBFβ) also binds to the RD to enhance RD–DNA interaction by 6- to 10-fold. Here, we report results from molecular dynamics (MD) simulations of RD alone, as a dimer in complexes with DNA and CBFβ and in a ternary complex with DNA and CBFβ. Consistent with the experimental findings, in the presence of CBFβ the estimated free energy of binding of RD to the DNA is more favorable, which is shown to be due to more favorable intermolecular interactions and desolvation contributions. Also contributing to the enhanced binding are favorable intramolecular interactions between the ‘wing’ residues (RD residues 139–145) and the ‘wing1’ residues (RD residues 104–116). The simulation studies also indicate that the RD–CBFβ binding is more favorable in the presence of DNA due to a more favorable RD–CBFβ interaction energy. In addition, it is predicted that long-range interactions involving ionic residues contribute to binding cooperativity. Results from the MD calculations are used to interpret a variety of experimental mutagenesis data. A novel role for RD Glu116 to the RD–CBFβ interaction is predicted.     

The thermodynamics of protein-ligand interaction and solvation: insights for ligand design

Isothermal titration calorimetry is able to provide accurate information on the thermodynamic contributions of enthalpy and entropy changes to free energies of binding. The Structure/Calorimetry of Reported Protein Interactions Online database of published isothermal titration calorimetry studies and structural information on the interactions between proteins and small-molecule ligands is used here to reveal general thermodynamic properties of protein-ligand interactions and to investigate correlations with changes in solvation. The overwhelming majority of interactions are found to be enthalpically favoured. Synthetic inhibitors and biological ligands form two distinct subpopulations in the data, with the former having greater average affinity due to more favourable entropy changes on binding. The greatest correlation is found between the binding free energy and apolar surface burial upon complex formation. However, the free-energy contribution per unit area buried is only 30-50% of that expected from earlier studies of transfer free energies of small molecules. A simple probability-based estimator for the maximal affinity of a binding site in terms of its apolar surface area is proposed. Polar surface area burial also contributes substantially to affinity but is difficult to express in terms of unit area due to the small variation in the amount of polar surface buried and a tendency for cancellation of its enthalpic and entropic contributions. Conventionally, the contribution of apolar desolvation to affinity is attributed to gain of entropy due to solvent release. Although data presented here are supportive of this notion, because the correlation of entropy change with apolar surface burial is relatively weak, it cannot, on present evidence, be confidently considered to be correct. Further, thermodynamic changes arising from small differences between ligands binding to individual proteins are relatively large and, in general, uncorrelated with changes in solvation, suggesting that trends identified across widely differing proteins are of limited use in explaining or predicting the effects of ligand modifications.     

Molecular dynamics simulations of the adipocyte lipid binding protein reveal a novel entry site for the ligand

The adipocyte lipid binding protein (ALBP) binds fatty acids (FA) in a cavity that is inaccessible from the bulk. Therefore, the penetration of the FA necessitates conformational changes whose nature is still unknown. It was suggested that the lipid first enters through a "portal region" which consists of the alphaII helix and the adjacent tight turns. The initial event in the ligand binding must be the interaction of the lipid with the protein surface. To analyze this interaction, we have carried out three molecular dynamics simulations of the apo-ALBP, with a palmitate ion initially located at different positions near the protein surface. The simulation indicated that the ligand could adsorb to the protein in more than one location. Yet, in one case, the ligand managed to penetrate the protein by entering a newly formed cavity some 10 A deep. The entry site is located near the N-terminus, at the junction between the loops connecting the beta-strands. Further progression of the penetration seems to be arrested by the need for desolvation of the COOH end of the headgroup. Evolutionary analysis showed that amino acids in this entry site are well conserved. On the basis of these observations, we suggest that the ligand may enter the protein from a site other than the portal region. Furthermore, the rate-limiting step is proposed to be the desolvation of the FA polar headgroup.