Insights from the energetics of water binding at the domain-ligand interface of the Src SH2 domain

SH2 domains play important roles in signal transduction by binding phosphorylated tyrosine residues on cell surface receptors. In an effort to understand the mechanism of ligand binding and more specifically the role of water, we have designed a general computational protocol based on the potential of mean force to compute the thermodynamics of water molecules at the protein-ligand interface for two SH2 domain complexes of the Src kinase, those bound to the two peptides Ac-PQpYEpYI-NH2 and Ac-PQpYIpYV-NH2 where pY indicates a phosphotyrosine. These two peptides were chosen because they have similar binding affinities but very different entropic/enthalpic thermodynamic binding signatures, indicating different interactions with solvent. We find that the isoleucine to valine mutation at position +3 (the third amino acid C-terminal to pY) in the ligand has only limited impact on the water structure. By contrast, the glutamic acid to isoleucine mutation at position +1 has a significant impact by not only abrogating a local hydrophilic binding site but, more importantly and surprisingly, inducing a favorable nonlocal entropic contribution from the water molecules around the phosphorylated tyrosine at the +2 position. Our study demonstrates the validity of the method reported here for exploring the thermodynamic solvation landscape of protein-protein interactions.     

Non-additivity of Functional Group Contributions in Protein-Ligand Binding: A Comprehensive Study by Crystallography and Isothermal Titration Calorimetry

Additivity of functional group contributions to protein-ligand binding is a very popular concept in medicinal chemistry as the basis of rational design and optimized lead structures. Most of the currently applied scoring functions for docking build on such additivity models. Even though the limitation of this concept is well known, case studies examining in detail why additivity fails at the molecular level are still very scarce. The present study shows, by use of crystal structure analysis and isothermal titration calorimetry for a congeneric series of thrombin inhibitors, that extensive cooperative effects between hydrophobic contacts and hydrogen bond formation are intimately coupled via dynamic properties of the formed complexes. The formation of optimal lipophilic contacts with the surface of the thrombin S3 pocket and the full desolvation of this pocket can conflict with the formation of an optimal hydrogen bond between ligand and protein. The mutual contributions of the competing interactions depend on the size of the ligand hydrophobic substituent and influence the residual mobility of ligand portions at the binding site. Analysis of the individual crystal structures and factorizing the free energy into enthalpy and entropy demonstrates that binding affinity of the ligands results from a mixture of enthalpic contributions from hydrogen bonding and hydrophobic contacts, and entropic considerations involving an increasing loss of residual mobility of the bound ligands. This complex picture of mutually competing and partially compensating enthalpic and entropic effects determines the non-additivity of free energy contributions to ligand binding at the molecular level.     

Long-Timescale Molecular-Dynamics Simulations of the Major Urinary Protein Provide Atomistic Interpretations of the Unusual Thermodynamics of Ligand Binding

The mouse major urinary protein (MUP) has proved to be an intriguing test bed for detailed studies on protein-ligand recognition. NMR, calorimetric, and modeling investigations have revealed that the thermodynamics of ligand binding involve a complex interplay between competing enthalpic and entropic terms. We performed six independent, 1.2 μs molecular-dynamics simulations on MUP—three replicates on the apo-protein, and three on the complex with the pheromone isobutylmethoxypyrazine. Our findings provide the most comprehensive picture to date of the structure and dynamics of MUP, and how they are modulated by ligand binding. The mechanical pathways by which amino acid side chains can transmit information regarding ligand binding to surface loops and either increase or decrease their flexibility (entropy-entropy compensation) are identified. Dewetting of the highly hydrophobic binding cavity is confirmed, and the results reveal an aspect of ligand binding that was not observed in earlier, shorter simulations: bound ligand retains extensive rotational freedom. Both of these features have significant implications for interpretations of the entropic component of binding. More generally, these simulations test the ability of current molecular simulation methods to produce a reliable and reproducible picture of protein dynamics on the microsecond timescale.     

Contributions of water transfer energy to protein‐ligand association and dissociation barriers: Watermap analysis of a series of p38α MAP kinase inhibitors

In our previous work, we proposed that desolvation and resolvation of the binding sites of proteins can serve as the slowest steps during ligand association and dissociation, respectively, and tested this hypothesis on two protein‐ligand systems with known binding kinetics behavior. In the present work, we test this hypothesis on another kinetically‐determined protein‐ligand system—that of p38α and eight Type II BIRB 796 inhibitor analogs. The k_on values among the inhibitor analogs are narrowly distributed (10⁴ ≤ k_on ≤ 10⁵ M^?1 s^?1), suggesting a common rate‐determining step, whereas the k_off values are widely distributed (10^?1 ≤ k_off ≤ 10^?6 s^?1), suggesting a spectrum of rate‐determining steps. We calculated the solvation properties of the DFG‐out protein conformation using an explicit solvent molecular dynamics simulation and thermodynamic analysis method implemented in WaterMap to predict the enthalpic and entropic costs of water transfer to and from bulk solvent incurred upon association and dissociation of each inhibitor. The results suggest that the rate‐determining step for association consists of the transfer of a common set of enthalpically favorable solvating water molecules from the binding site to bulk solvent. The rate‐determining step for inhibitor dissociation consists of the transfer of water from bulk solvent to specific binding site positions that are unfavorably solvated in the apo protein, and evacuated during ligand association. Different sets of unfavorable solvation are evacuated by each ligand, and the observed dissociation barriers are qualitatively consistent with the calculated solvation free energies of those sets.     

Coupling of global and local vibrational modes in dynamic allostery of proteins

It is now recognized that internal global protein dynamics play an important role in the allosteric function of many proteins. Alterations of protein flexibility on effector binding affect the entropic cost of binding at a distant site. We present a coarse-grained model for a potential amplification of such entropic allostery due to coupling of fast, localized modes to the slow, global modes. We show how such coupling can give rise to large compensating entropic and enthalpic terms. The model corresponds to the pattern of calorimetry and NMR data from experiments on the Met repressor.     

Structural and thermodynamic studies of binding saturated fatty acids to bovine β-lactoglobulin

Lactoglobulin is a globular milk protein for which physiological function has not been clarified. Due to its binding properties lactoglobulin might serve as a carrier for bioactive molecules. Binding of 12-, 14-, 16- and 18-carbon saturated fatty acids to bovine β-lactoglobulin has been characterised by isothermal titration calorimetry and X-ray crystallography as a part of systematic studies of lactoglobulin complexes with ligands of biological importance. The thermodynamic parameters have been determined for lauric, myristic and palmitic acid complexes revealing systematic decrease of enthalpic and increase of entropic component of ΔG with elongation of aliphatic chain. In all crystal structures determined with resolution 1.9-2.1?, single fatty acid molecule was found in the β-barrel in extended conformation with individual pattern of interactions. Location of a fatty acid in the binding site depends on the length of aliphatic chain and influences polar interactions between protein and ligand. Systematic changes of entropic component indicate important role of water in binding process.     

Microcalorimetric indications for ligand binding as a function of the protein for galactoside-specific plant and avian lectins

The process cascade leading to the final accommodation of the carbohydrate ligand in the lectin's binding site comprises enthalpic and entropic contributions of the binding partners and solvent molecules. With emphasis on lactose, N-acetyllactosamine, and thiodigalactoside as potent inhibitors of binding of galactoside-specific lectins, the question was addressed to what extent these parameters are affected as a function of the protein. The microcalorimetric study of carbohydrate association to the galectin from chicken liver (CG-16) and the agglutinin from Viscum album (VAA) revealed enthalpy-entropy compensation with evident protein type-dependent changes for N-acetyllactosamine. Reduction of the entropic penalty by differential flexibility of loops or side chains and/or solvation properties of the protein will have to be reckoned with to assign a molecular cause to protein type-dependent changes in thermodynamic parameters for lectins sharing the same monosaccharide specificity.     

Thermodynamics of metal ion binding. 2. Metal ion binding by carbonic anhydrase variants

The ability to construct molecular motifs with predictable properties in aqueous solution requires an extensive knowledge of the relationships between structure and energetics. The design of metal binding motifs is currently an area of intense interest in the bioorganic community. To date synthetic motifs designed to bind metal ions lack the remarkable affinities observed in biological systems. To better understand the structural basis of metal ion affinity, we report here the thermodynamics of binding of divalent zinc ions to wild-type and mutant carbonic anhydrases and the interpretation of these parameters in terms of structure. Mutations were made both to the direct His ligand at position 94 and to indirect, or second-shell, ligands Gln-92, Glu-117, and Thr-199. The thermodynamics of ligand binding by several mutant proteins is complicated by the development of a second zinc binding site on mutation; such effects must be considered carefully in the interpretation of thermodynamic data. In all instances modification of the protein produces a complex series of changes in both the enthalpy and entropy of ligand binding. In most cases these effects are most readily rationalized in terms of ligand and protein desolvation, rather than in terms of changes in the direct interactions of ligand and protein. Alteration of second-shell ligands, thought to function primarily by orienting the direct ligands, produces profoundly different effects on the enthalpy of binding, depending on the nature of the residue. These results suggest a range of activities for these ligands, contributing both enthalpic and entropic effects to the overall thermodynamics of binding. Together, our results demonstrate the importance of understanding relationships between structure and hydration in the construction of novel ligands and biological polymers.     

Streptavidin-biotin binding energetics

The high affinity energetics in the streptavidin-biotin system provide an excellent model system for studying how proteins balance enthalpic and entropic components to generate an impressive overall free energy for ligand binding. We review here concerted site-directed mutagenesis, biophysical, and computational studies of aromatic and hydrogen bonding interaction energetics between streptavidin and biotin. These results also have provided insight into how streptavidin builds a large activation barrier to dissociation by managing the enthalpic and entropic activation components. Finally, we review recent studies of the biotin dissociation pathway that address the fundamental question of how ligands exit protein binding pockets.     

Heat capacity effects of water molecules and ions at a protein-DNA interface

The interaction of the TATA-box binding protein from the thermophilic and halophilic archaea Pyrococcus woesei (PwTBP) with an oligonucleotide containing a specific binding site is stable over a very broad range of temperatures and ionic strengths, and is consequently an outstanding system for characterising general features of protein-DNA thermodynamics. In common with other specific protein-DNA recognition events, the PwTBP-TATA box interaction is accompanied by a large negative change in heat capacity (ΔC_p) arising from the total change in solvation that occurs upon binding, which in this case involves a net uptake of cations. Contrary to previous hypotheses, we find no overall effect of ionic strength on this heat capacity change. We investigate the local contributions of site-specific ion and water binding to the overall change in heat capacity by means of a series of site-directed mutations of PwTBP. We find that although changes in the local ion binding capacity affect the enthalpic and entropic contributions to the free energy of the interaction, they do not affect the change in heat capacity. In contrast, we find remarkably large heat capacity effects arising from two particular symmetry-related mutations. The great magnitude of these effects is not explicable in terms of current semi-empirical models of heat capacity change. Previously reported X-ray crystal structures show that these mutated residues are at the centre of an evolutionarily conserved network of water-mediated hydrogen bonds between the protein and the DNA backbone. Consequently, we conclude that, in addition to water molecules buried in the protein-DNA interface that have been previously shown to influence heat capacity, bridging water molecules in a highly polar surface environment can also contribute substantially to negative heat capacity change on formation of a protein-DNA complex.     

Enthalpy screen of drug candidates

The enthalpic and entropic contributions to the binding affinity of drug candidates have been acknowledged to be important determinants of the quality of a drug molecule. These quantities, usually summarized in the thermodynamic signature, provide a rapid assessment of the forces that drive the binding of a ligand. Having access to the thermodynamic signature in the early stages of the drug discovery process will provide critical information towards the selection of the best drug candidates for development. In this paper, the Enthalpy Screen technique is presented. The enthalpy screen allows fast and accurate determination of the binding enthalpy for hundreds of ligands. As such, it appears to be ideally suited to aid in the ranking of the hundreds of hits that are usually identified after standard high throughput screening.     

Free energetics of rigid body association of ubiquitin binding domains: A biochemical model for binding mediated by hydrophobic interaction

Weak intermolecular interactions, such as hydrophobic associations, underlie numerous biomolecular recognition processes. Ubiquitin is a small protein that represents a biochemical model for exploring thermodynamic signatures of hydrophobic association as it is widely held that a major component of ubiquitin's binding to numerous partners is mediated by hydrophobic regions on both partners. Here, we use atomistic molecular dynamics simulations in conjunction with the Adaptive Biasing Force sampling method to compute potentials of mean force (the reversible work, or free energy, associated with the binding process) to investigate the thermodynamic signature of complexation in this well‐studied biochemical model of hydrophobic association. We observe that much like in the case of a purely hydrophobic solute (i.e., graphene, carbon nanotubes), association is favored by entropic contributions from release of water from the interprotein regions. Moreover, association is disfavored by loss of enthalpic interactions, but unlike in the case of purely hydrophobic solutes, in this case protein‐water interactions are lost and not compensated for by additional water‐water interactions generated upon release of interprotein and moreso, hydration, water. We further find that relative orientations of the proteins that mutually present hydrophobic regions of each protein to its partner are favored over those that do not. In fact, the free energy minimum as predicted by a force field based method recapitulates the experimental NMR solution structure of the complex. Proteins 2014; 82:1453–1468. © 2014 Wiley Periodicals, Inc.     

Studies on the binding sites of IgG2 monoclonal antibodies recognized by terpyridine‐based affinity ligands

This investigation has examined the origin of the molecular recognition associated with the interaction of monoclonal IgG2's with terpyridine‐based ligands immobilized onto agarose‐derived chromatographic adsorbents. Isothermal titration calorimetric (ITC) methods have been employed to acquire thermodynamic data associated with the IgG2‐ligand binding. These ITC investigations have documented that different enthalpic and entropic processes are involved depending on the nature of the chemical substituents in the core structure of the terpyridinyl moiety. In addition, molecular docking studies have been carried out with IgG2 structures with the objective to identify possible ligand binding sites and key interacting amino acid residues. These molecular docking experiments with the different terpyridine‐based ligands have shown that all of the examined ligands can potentially undergo favorable interactions with a site located within the Fab region of the IgG2. However, another favorable binding site was also identified from the docking poses to exist within the Fc region of the IgG2 for some, but not all, of the ligands studied. These investigations have provided a basis to elucidate the unique binding properties and chromatographic behaviors shown by several substituted terpyridine ligands in their interaction with IgGs of different isotype. Copyright © 2016 John Wiley & Sons, Ltd.     

Not only enthalpy: large entropy contribution to ion permeation barriers in single-file channels

The effect of channel length on the barrier for potassium ion permeation through single-file channels has been studied by means of all-atom molecular dynamics simulations. Using series of peptidic gramicidin-like and simplified ring-structured channels, both embedded in model membranes, we obtained two distinct types of behavior: saturation of the central free energy barriers for peptidic channels and a linear increase in simplified ring-structured channels with increasing channel length. The saturation of the central free energy barrier for the peptidic channels occurs at relatively short lengths, and it is correlated with the desolvation from the bulk water. Remarkably, decomposition of free energy barriers into enthalpic and entropic terms reveals an entropic cost for ion permeation. Furthermore, this entropic cost dominates the ion permeation free energy barrier, since the corresponding free energy contribution is higher than the enthalpic barrier. We conclude that the length dependence of the free energy is enthalpy-dominated, but the entropy is the major contribution to the permeation barrier. The decrease in rotational water motion and the reduction of channel mobility are putative origins for the overall entropic penalty.     

Global changes in local protein dynamics reduce the entropic cost of carbohydrate binding in the arabinose-binding protein

Protein dynamics make important but poorly understood contributions to molecular recognition phenomena. To address this, we measure changes in fast protein dynamics that accompany the interaction of the arabinose-binding protein (ABP) with its ligand, d-galactose, using NMR relaxation and molecular dynamics simulation. These two approaches present an entirely consistent view of the dynamic changes that occur in the protein backbone upon ligand binding. Increases in the amplitude of motions are observed throughout the protein, with the exception of a few residues in the binding site, which show restriction of dynamics. These counter-intuitive results imply that a localised binding event causes a global increase in the extent of protein dynamics on the pico- to nanosecond timescale. This global dynamic change constitutes a substantial favourable entropic contribution to the free energy of ligand binding. These results suggest that the structure and dynamics of ABP may be adapted to exploit dynamic changes to reduce the entropic costs of binding.     

Thermodynamic dissection of the binding energetics of proline-rich peptides to the Abl-SH3 domain: implications for rational ligand design

The inhibition of the interactions between SH3 domains and their targets is emerging as a promising therapeutic strategy. To date, rational design of potent ligands for these domains has been hindered by the lack of understanding of the origins of the binding energy. We present here a complete thermodynamic analysis of the binding energetics of the p41 proline-rich decapeptide (APSYSPPPPP) to the SH3 domain of the c-Abl oncogene. Isothermal titration calorimetry experiments have revealed a thermodynamic signature for this interaction (very favourable enthalpic contributions opposed by an unfavourable binding entropy) inconsistent with the highly hydrophobic nature of the p41 ligand and the Abl-SH3 binding site. Our structural and thermodynamic analyses have led us to the conclusion, having once ruled out any possible ionization events or conformational changes coupled to the association, that the establishment of a complex hydrogen-bond network mediated by water molecules buried at the binding interface is responsible for the observed thermodynamic behaviour. The origin of the binding energetics for proline-rich ligands to the Abl-SH3 domain is further investigated by a comparative calorimetric analysis of a set of p41-related ligands. The striking effects upon the enthalpic and entropic contributions provoked by conservative substitutions at solvent-exposed positions in the ligand confirm the complexity of the interaction. The implications of these results for rational ligand design are discussed.     

Thermodynamic analysis of the equilibrium, association and dissociation of 2'GMP and 3'GMP with ribonuclease T1 at pH 5.3.

Fluorescence titrations and temperature-jump relaxation experiments were performed as a function of temperature on ribonuclease T1 with the inhibitors 2'GMP and 3'GMP to obtain information on the energetics and molecular events controlling the binding of those inhibitors. Results from the titration and temperature-jump experiments were in agreement concerning the equilibrium constant. The larger equilibrium constant for 2'GMP is enthalpic in origin and is due to both a higher on rate and a lower off rate as compared to 3'GMP. On rates for both inhibitors appear to be below the diffusion controlled limit, apparently due to conformational changes in the portion of the active site responsible for recognition of the guanine base. Comparison of the measured enthalpic and entropic terms associated with the equilibrium constant determined from the fluorescence titrations are in disagreement with those calculated from the on and off rates indicating the presence of an induced conformational change in the 2'GMP-enzyme complex. This second conformational change appears to be due to additional interactions between 2'GMP and the catalytic portion of the active site, which may also be responsible for the differences in the binding constant, the on rate and the off rate between 2'GMP and 3'GMP.     

Mapping the energetics of water-protein and water-ligand interactions with the "natural" HINT forcefield: predictive tools for characterizing the roles of water in biomolecules

The energetics and hydrogen bonding pattern of water molecules bound to proteins were mapped by analyzing structural data (resolution better than 2.3A) for sets of uncomplexed and ligand-complexed proteins. Water-protein and water-ligand interactions were evaluated using hydropatic interactions (HINT), a non-Newtonian forcefield based on experimentally determined logP(octanol/water) values. Potential water hydrogen bonding ability was assessed by a new Rank algorithm. The HINT-derived binding energies and Ranks for second shell water molecules were -0.04 kcal mol(-1) and 0.0, respectively, for first shell water molecules -0.38 kcal mol(-1) and 1.6, for active site water molecules -0.45 kcal mol(-1) and 2.3, for cavity water molecules -0.55 kcal mol(-1) and 3.3, and for buried water molecules -0.56 kcal mol(-1) and 4.4. For the last four classes, similar energies indicate that internal and external water molecules interact with protein almost equally, despite different degrees of hydrogen bonding. The binding energies and Ranks for water molecules bridging ligand-protein were -1.13 kcal mol(-1) and 4.5, respectively. This energetic contribution is shared equally between protein and ligand, whereas Rank favors the protein. Lastly, by comparing the uncomplexed and complexed forms of proteins, guidelines were developed for prediction of the roles played by active site water molecules in ligand binding. A water molecule with high Rank and HINT score is unlikely to make further interactions with the ligand and is largely irrelevant to the binding process, while a water molecule with moderate Rank and high HINT score is available for ligand interaction. Water molecule displaced for steric reasons were characterized by lower Rank and HINT score. These guidelines, tested by calculating HINT score and Rank for 50 water molecules bound in the active site of four uncomplexed proteins (for which the structures of the liganded forms were also available), correctly predicted the ultimate roles (in the complex) for 76% of water molecules. Some failures were likely due to ambiguities in the structural data.     

Temperature and pressure adaptation of the binding site of acetylcholinesterase

1. Studies with a carbon substrate analogue, 3,3-dimethylbutyl acetate, indicate that the hydrophobic contribution to binding at the anionic site of acetylcholinesterase is strongly disrupted at low temperatures and high pressures. 2. Animals living in different physical environments circumvent this problem by adjusting the enthalpic and entropic contributions to binding. 3. An extreme example of this adaptational strategy is supplied by brain acetylcholinesterase extracted from an abyssal fish living at 2 degrees C and up to several hundred atmospheres of pressure. This acetylcholinesterase appears to have a smaller hydrophobic binding region in the anionic site, playing a measurably decreased role in ligand binding.