Thermodynamic Properties Of Water Molecules Research Articles

WaterKit: Thermodynamic Profiling of Protein Hydration Sites.

Water desolvation is one of the key components of the free energy of binding of small molecules to their receptors. Thus, understanding the energetic balance of solvation and desolvation resulting from individual water molecules can be crucial when estimating ligand binding, especially when evaluating different molecules and poses as done in High-Throughput Virtual Screening (HTVS). Over the most recent decades, several methods were developed to tackle this problem, ranging from fast approximate methods (usually empirical functions using either discrete atom-atom pairwise interactions or continuum solvent models) to more computationally expensive and accurate ones, mostly based on Molecular Dynamics (MD) simulations, such as Grid Inhomogeneous Solvation Theory (GIST) or Double Decoupling. On one hand, MD-based methods are prohibitive to use in HTVS to estimate the role of waters on the fly for each ligand. On the other hand, fast and approximate methods show an unsatisfactory level of accuracy, with low agreement with results obtained with the more expensive methods. Here we introduce WaterKit, a new grid-based sampling method with explicit water molecules to calculate thermodynamic properties using the GIST method. Our results show that the discrete placement of water molecules is successful in reproducing the position of crystallographic waters with very high accuracy, as well as providing thermodynamic estimates with accuracy comparable to more expensive MD simulations. Unlike these methods, WaterKit can be used to analyze specific regions on the protein surface, (such as the binding site of a receptor), without having to hydrate and simulate the whole receptor structure. The results show the feasibility of a general and fast method to compute thermodynamic properties of water molecules, making it well-suited to be integrated in high-throughput pipelines such as molecular docking.

Potency-Enhancing Mutations of Gating Modifier Toxins for the Voltage-Gated Sodium Channel NaV1.7 Can Be Predicted Using Accurate Free-Energy Calculations.

Gating modifier toxins (GMTs) isolated from venomous organisms such as Protoxin-II (ProTx-II) and Huwentoxin-IV (HwTx-IV) that inhibit the voltage-gated sodium channel NaV1.7 by binding to its voltage-sensing domain II (VSDII) have been extensively investigated as non-opioid analgesics. However, reliably predicting how a mutation to a GMT will affect its potency for NaV1.7 has been challenging. Here, we hypothesize that structure-based computational methods can be used to predict such changes. We employ free-energy perturbation (FEP), a physics-based simulation method for predicting the relative binding free energy (RBFE) between molecules, and the cryo electron microscopy (cryo-EM) structures of ProTx-II and HwTx-IV bound to VSDII of NaV1.7 to re-predict the relative potencies of forty-seven point mutants of these GMTs for NaV1.7. First, FEP predicted these relative potencies with an overall root mean square error (RMSE) of 1.0 ± 0.1 kcal/mol and an R2 value of 0.66, equivalent to experimental uncertainty and an improvement over the widely used molecular-mechanics/generalized born-surface area (MM-GB/SA) RBFE method that had an RMSE of 3.9 ± 0.8 kcal/mol. Second, inclusion of an explicit membrane model was needed for the GMTs to maintain stable binding poses during the FEP simulations. Third, MM-GB/SA and FEP were used to identify fifteen non-standard tryptophan mutants at ProTx-II[W24] predicted in silico to have a at least a 1 kcal/mol gain in potency. These predicted potency gains are likely due to the displacement of high-energy waters as identified by the WaterMap algorithm for calculating the positions and thermodynamic properties of water molecules in protein binding sites. Our results expand the domain of applicability of FEP and set the stage for its prospective use in biologics drug discovery programs involving GMTs and NaV1.7.

Water Thermodynamics of Peptide Toxin Binding Sites on Ion Channels.

Peptide toxins isolated from venomous creatures, long prized as research tools due to their innate potency for ion channels, are emerging as drugs as well. However, it remains challenging to understand why peptide toxins bind with high potency to ion channels, to identify residues that are key for activity, and to improve their affinities via mutagenesis. We use WaterMap, a molecular dynamics simulation-based method, to gain computational insight into these three questions by calculating the locations and thermodynamic properties of water molecules in the peptide toxin binding sites of five ion channels. These include an acid-sensing ion channel, voltage-gated potassium channel, sodium channel in activated and deactivated states, transient-receptor potential channel, and a nicotinic receptor whose structures were recently determined by crystallography and cryo-electron microscopy (cryo-EM). All channels had water sites in the peptide toxin binding site, and an average of 75% of these sites were stable (low-energy), and 25% were unstable (medium or high energy). For the sodium channel, more unstable water sites were present in the deactivated state structure than the activated. Additionally, for each channel, unstable water sites coincided with the positions of peptide toxin residues that previous mutagenesis experiments had shown were important for activity. Finally, for the sodium channel in the deactivated state, unstable water sites were present in the peptide toxin binding pocket but did not overlap with the peptide toxin, suggesting that future experimental efforts could focus on targeting these sites to optimize potency.

Solvation Free Energy as a Measure of Hydrophobicity: Application to Serine Protease Binding Interfaces.

Solvation and hydrophobicity play a key role in a variety of biological mechanisms. In substrate binding, but also in structure-based drug design, the thermodynamic properties of water molecules surrounding a given protein are of high interest. One of the main algorithms devised in recent years to quantify thermodynamic properties of water is the grid inhomogeneous solvation theory (GIST), which calculates these features on a grid surrounding the protein. Despite the inherent advantages of GIST, the computational demand is a major drawback, as calculations for larger systems can take days or even weeks. Here, we present a GPU accelerated version of the GIST algorithm, which facilitates efficient estimates of solvation free energy even of large biomolecular interfaces. Furthermore, we show that GIST can be used as a reliable tool to evaluate protein surface hydrophobicity. We apply the approach on a set of nine different proteases calculating localized solvation free energies on the surface of the binding interfaces as a measure of their hydrophobicity. We find a compelling agreement with the hydrophobicity of their substrates, i.e., peptides, binding into the binding cleft, and thus our approach provides a reliable description of hydrophobicity characteristics of these biological interfaces.

PyRod: Tracing Water Molecules in Molecular Dynamics Simulations.

Ligands entering a protein binding pocket essentially compete with water molecules for binding to the protein. Hence, the location and thermodynamic properties of water molecules in protein structures have gained increased attention in the drug design community. Including corresponding data into 3D pharmacophore modeling is essential for efficient high throughput virtual screening. Here, we present PyRod, a free and open-source Python software that allows for visualization of pharmacophoric binding pocket characteristics, identification of hot spots for ligand binding, and subsequent generation of pharmacophore features for virtual screening. The implemented routines analyze the protein environment of water molecules in molecular dynamics (MD) simulations and can differentiate between hydrogen bonded waters as well as waters in a protein environment of hydrophobic, charged, or aromatic atom groups. The gathered information is further processed to generate dynamic molecular interaction fields (dMIFs) for visualization and pharmacophoric features for virtual screening. The described software was applied to 5 therapeutically relevant drug targets, and generated pharmacophores were evaluated using DUD-E benchmarking sets. The best performing pharmacophore was found for the HIV1 protease with an early enrichment factor of 54.6. PyRod adds a new perspective to structure-based screening campaigns by providing easy-to-interpret dMIFs and purely protein-based pharmacophores that are solely based on tracing water molecules in MD simulations. Since structural information about cocrystallized ligands is not needed, screening campaigns can be followed, for which less or no ligand information is available. PyRod is freely available at https://github.com/schallerdavid/pyrod .

Calculation of Thermodynamic Properties of Bound Water Molecules.

Water molecules in the binding site of a protein significantly influence protein structure and function, for example, by mediating protein-ligand interactions or in form of desolvation as driving force for ligand binding. The knowledge about location and thermodynamic properties of water molecules in the binding site is crucial to the understanding of protein function. This chapter describes the method of calculating the location and thermodynamic properties of bound water molecules from molecular dynamics (MD) simulation trajectories. Thermodynamic profiles of water molecules can be calculated either with or without the presence of a bound ligand based on the scientific problem. The location and thermodynamic profile of hydration sites mediating the protein-ligand interactions is important for understanding protein-ligand binding. The protein desolvation free energy can be estimated for any ligand by summation of the hydration site free energies of the displaced hydration sites. The WATsite program with an easy-to-use graphical user interface (GUI) based on PyMOL was developed for those calculations and is discussed in this chapter. The WATsite program and its PyMOL plugin are available free of charge from http://people.pharmacy.purdue.edu/~mlill/software/watsite/version3.shtml .

Local thermodynamics of the water molecules around single- and double-stranded DNA studied by grid inhomogeneous solvation theory

Thermodynamic properties of water molecules around single- and double-stranded DNAs (ssDNAs and dsDNAs) with different sequences were investigated using grid inhomogeneous solvation theory. Free energies of water molecules solvating the minor groove of dsDNAs are lower than those near ssDNAs, while water molecules should be released during the formation of dsDNA. Free energies of water molecules around dsDNA are lower than those around ssDNA even in the second and third hydration shells. Our findings will help to clarify the role of water molecules in the formation of dsDNA from ssDNAs, thus facilitating the designs of drugs or nanomaterials using DNA.

Thermodynamic properties of water molecules in the presence of cosolute depend on DNA structure: a study using grid inhomogeneous solvation theory

In conditions that mimic those of the living cell, where various biomolecules and other components are present, DNA strands can adopt many structures in addition to the canonical B-form duplex. Previous studies in the presence of cosolutes that induce molecular crowding showed that thermal stabilities of DNA structures are associated with the properties of the water molecules around the DNAs. To understand how cosolutes, such as ethylene glycol, affect the thermal stability of DNA structures, we investigated the thermodynamic properties of water molecules around a hairpin duplex and a G-quadruplex using grid inhomogeneous solvation theory (GIST) with or without cosolutes. Our analysis indicated that (i) cosolutes increased the free energy of water molecules around DNA by disrupting water–water interactions, (ii) ethylene glycol more effectively disrupted water–water interactions around Watson–Crick base pairs than those around G-quartets or non-paired bases, (iii) due to the negative electrostatic potential there was a thicker hydration shell around G-quartets than around Watson–Crick-paired bases. Our findings suggest that the thermal stability of the hydration shell around DNAs is one factor that affects the thermal stabilities of DNA structures under the crowding conditions.

Structure, dynamics and thermodynamics of single-file water under confinement: effects of polarizability of water molecules

Various structural, dynamic and thermodynamic properties of water molecules confined in single-wall carbon nanotubes are investigated using both polarizable and non-polarizable water models.

Evaluation of Host-Guest Binding Thermodynamics of Model Cavities with Grid Cell Theory.

A previously developed cell theory model of liquid water was used to evaluate the excess thermodynamic properties of confined clusters of water molecules. The results are in good agreement with reference thermodynamic integration calculations, suggesting that the model is adequate to probe the thermodynamic properties of water at interfaces or in cavities. Next, the grid cell theory (GCT) method was applied to elucidate the thermodynamic signature of nonpolar association for a range of idealized host-guest systems. Polarity and geometry of the host cavities were systematically varied, and enthalpic and entropic solvent components were spatially resolved for detailed graphical analyses. Perturbations in the thermodynamic properties of water molecules upon guest binding are restricted to the immediate vicinity of the guest in solvent-exposed cavities, whereas longer-ranged perturbations are observed in buried cavities. Depending on the polarity and geometry of the host, water displacement by a nonpolar guest makes a small or large enthalpic or entropic contribution to the free energy of binding. Thus, no assumptions about the thermodynamic signature of the hydrophobic effect can be made in general. Overall the results warrant further applications of GCT to more complex systems such as protein-ligand complexes.

Water Network Perturbation in Ligand Binding: Adenosine A2AAntagonists as a Case Study

Recent efforts in the computational evaluation of the thermodynamic properties of water molecules have resulted in the development of promising new in silico methods to evaluate the role of water in ligand binding. These methods include WaterMap, SZMAP, GRID/CRY probe, and Grand Canonical Monte Carlo simulations. They allow the prediction of the position and relative free energy of the water molecule in the protein active site and the analysis of the perturbation of an explicit water network (WNP) as a consequence of ligand binding. We have for the first time extended these approaches toward the prediction of kinetics for small molecules and of relative free energy of binding with a focus on the perturbation of the water network and application to large diverse data sets. Our results support a qualitative correlation between the residence time of 12 related triazine adenosine A(2A) receptor antagonists and the number and position of high energy trapped solvent molecules. From a quantitative viewpoint, we successfully applied these computational techniques as an implicit solvent alternative, in linear combination with a molecular mechanics force field, to predict the relative ligand free energy of binding (WNP-MMSA). The applicability of this linear method, based on the thermodynamics additivity principle, did not extend to 375 diverse A(2A) receptor antagonists. However, a fast but effective method could be enabled by replacing the linear approach with a machine learning technique using probabilistic classification trees, which classified the binding affinity correctly for 90% of the ligands in the training set and 67% in the test set.

Benchmarking the thermodynamic analysis of water molecules around a model beta sheet.

Water molecules play a vital role in biological and engineered systems by controlling intermolecular interactions in the aqueous phase. Inhomogeneous fluid solvation theory provides a method to quantify solvent thermodynamics from molecular dynamics or Monte Carlo simulations and provides an insight into intermolecular interactions. In this study, simulations of TIP4P-2005 and TIP5P-Ewald water molecules around a model beta sheet are used to investigate the orientational correlations and predicted thermodynamic properties of water molecules at a protein surface. This allows the method to be benchmarked and provides information about the effect of a protein on the thermodynamics of nearby water molecules. The results show that the enthalpy converges with relatively little sampling, but the entropy and thus the free energy require considerably more sampling to converge. The two water models yield a very similar pattern of hydration sites, and these hydration sites have very similar thermodynamic properties, despite notable differences in their orientational preferences. The results also predict that a protein surface affects the free energy of water molecules to a distance of approximately 4.0 Å, which is in line with previous work. In addition, all hydration sites have a favorable free energy with respect to bulk water, but only when the water-water entropy term is included. A new technique for calculating this term is presented and its use is expected to be very important in accurately calculating solvent thermodynamics for quantitative application.

Thermodynamic analysis of water molecules at the surface of proteins and applications to binding site prediction and characterization

Water plays an essential role in determining the structure and function of all biological systems. Recent methodological advances allow for an accurate and efficient estimation of the thermodynamic properties of water molecules at the surface of proteins. In this work, we characterize these thermodynamic properties and relate them to various structural and functional characteristics of the protein. We find that high-energy hydration sites often exist near protein motifs typically characterized as hydrophilic, such as backbone amide groups. We also find that waters around alpha helices and beta sheets tend to be less stable than waters around loops. Furthermore, we find no significant correlation between the hydration site-free energy and the solvent accessible surface area of the site. In addition, we find that the distribution of high-energy hydration sites on the protein surface can be used to identify the location of binding sites and that binding sites of druggable targets tend to have a greater density of thermodynamically unstable hydration sites. Using this information, we characterize the FKBP12 protein and show good agreement between fragment screening hit rates from NMR spectroscopy and hydration site energetics. Finally, we show that water molecules observed in crystal structures are less stable on average than bulk water as a consequence of the high degree of spatial localization, thereby resulting in a significant loss in entropy. These findings should help to better understand the characteristics of waters at the surface of proteins and are expected to lead to insights that can guide structure-based drug design efforts.

Thermodynamic Properties of Water Molecules at a Protein–Protein Interaction Surface

Protein–protein interactions (PPIs) have been identified as a vital regulator of cellular pathways and networks. However, the determinants that control binding affinity and specificity at protein surfaces are incompletely characterized and thus unable to be exploited for the purpose of developing PPI inhibitors to control cellular pathways in disease states. One of the key factors in intermolecular interactions that remains poorly understood is the role of water molecules and in particular the importance of solvent entropy. This factor is expected to be particularly important at protein surfaces, and the release of water molecules from hydrophobic regions is one of the most important drivers of PPIs. In this work, we have studied the protein surface of a mutant of the protein RadA to quantify the thermodynamics of surface water molecules. RadA and its human homologue RAD51 function as recombinases in the process of homologous recombination. RadA binds to itself to form oligomeric structures and thus contains a well-characterized protein–protein binding surface. Similarly, RAD51 binds either to itself to form oligomers or to the protein BRCA2 to form filaments. X-ray crystallography has determined that the same interface functions in both interactions. Work in our group has generated a partially humanized mutant of RadA, termed MAYM, which has been crystallized in the apo form. We studied this apo form of MAYM using a combination of molecular dynamics (MD) simulations and inhomogeneous fluid solvation theory (IFST). The method locates a number of the hydration sites observed in the crystal structure and locates hydrophobic sites where hydrophobic species are known to bind experimentally. The simulations also highlight the importance of the restraints placed on the protein in determining the results. Finally, the results identify a correlation between the predicted entropy of water molecules at a given site and the solvent-accessible surface area and suggest that correlations between water molecules only need to be considered for water molecules separated by less than 3.2 Å. The combination of MD and IFST has been used previously to study PPIs and represents one of the few existing methods to quantify solvent thermodynamics. This is a vital aspect of molecular recognition and one which we believe must be developed.

Understanding Kinase Selectivity Through Energetic Analysis of Binding Site Waters

Kinases remain an important drug target class within the pharmaceutical industry; however, the rational design of kinase inhibitors is plagued by the complexity of gaining selectivity for a small number of proteins within a family of more than 500 related enzymes. Herein we show how a computational method for identifying the location and thermodynamic properties of water molecules within a protein binding site can yield insight into previously inexplicable selectivity and structure-activity relationships. Four kinase systems (Src family, Abl/c-Kit, Syk/ZAP-70, and CDK2/4) were investigated, and differences in predicted water molecule locations and energetics were able to explain the experimentally observed binding selectivity profiles. The successful predictions across the range of kinases studied here suggest that this methodology could be generally applicable for predicting selectivity profiles in related targets.

Carbohydrate-Binding Proteins: Dissecting Ligand Structures through Solvent Environment Occupancy

Formation of protein ligand complexes is a fundamental phenomenon in biochemistry. During the process, significant solvent reorganization is produced along the contact surface and many water molecules strongly bound to the protein's ligand binding site must be displaced. Both the thermodynamics and kinetics of this process are complex and a clear understanding at the microscopic level has been not achieved so far. Special attention has been paid to the structure of water molecules on carbohydrate recognition sites of various proteins, and many studies support the idea that displacement of these water molecules should have a crucial effect on the binding free energy. Molecular dynamics (MD) simulations in explicit water solvent is a very promising approach for this type of studies. Using MD simulations combined with statistical mechanics analysis, thermodynamic properties of these water molecules can be computed and analyzed in a comparative view. Using this idea, we developed a set of analysis tools to link solvation with ligand binding in a key carbohydrate binding protein, human galectin-1 (hGal-1). Specifically, we defined water sites (WS) in terms of the thermodynamic properties of water molecules strongly bound to protein surfaces. In the present work, we selected a group of proteins whose ligand bound complexes have been already structurally characterized in order to extend the analysis of the role of the surface associated water molecules in the ligand binding and recognition process. The selected proteins are concanavalin-A (Con-A), galectin-3 (Gal-3), cyclophilin-A (Cyp-A), and two modules CBM40 and CBM32 of the multimodular bacterial sialidase. Our results show that the probability of finding water molecules inside the WS, p(v), with respect to the bulk density is directly correlated to the likeliness of finding an hydroxyl group of the ligand in the protein-ligand complex. This information can be used to analyze in detail the solvation structure of the carbohydrate recognition domain (CRD) and its relation to the possible protein ligand complexes and suggests addition of OH-containing functional groups to displace water from high p(v) WS to enhance drugs, specially glycomimetic-drugs, protein affinity, and/or specificity.