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.     

Characterizing hydration sites in protein-ligand complexes towards the design of novel ligands

Water is an essential part of protein binding sites and mediates interactions to ligands. Its displacement by ligand parts affects the free binding energy of resulting protein-ligand complexes. Therefore the characterization of solvation properties is important for design. Of particular interest is the propensity of localized water to be favorably displaced by a ligand. This review discusses two popular computational approaches addressing these questions, namely WaterMap based on statistical mechanics analysis of MD simulations and 3D RISM based on integral equation theory of liquids. The theoretical background and recent applications in structure-based design will be presented.     

High‐energy water sites determine peptide binding affinity and specificity of PDZ domains

PDZ domains have well known binding preferences for distinct C‐terminal peptide motifs. For most PDZ domains, these motifs are of the form [S/T]‐W‐[I/L/V]. Although the preference for S/T has been explained by a specific hydrogen bond interaction with a histidine in the PDZ domain and the (I/L/V) is buried in a hydrophobic pocket, the mechanism for Trp specificity at the second to last position has thus far remained unknown. Here, we apply a method to compute the free energies of explicit water molecules and predict that potency gained by Trp binding is due to a favorable release of high‐energy water molecules into bulk. The affinities of a series of peptides for both wild‐type and mutant forms of the PDZ domain of Erbin correlate very well with the computed free energy of binding of displaced waters, suggesting a direct relationship between water displacement and peptide affinity. Finally, we show a correlation between the magnitude of the displaced water free energy and the degree of Trp‐sensitivity among subtypes of the HTRA PDZ family, indicating a water‐mediated mechanism for specificity of peptide binding.     

The translocation kinetics of antibiotics through porin OmpC: Insights from structure‐based solvation mapping using WaterMap

Poor permeability of the lipopolysaccharide‐based outer membrane of Gram‐negative bacteria is compensated by the existence of protein channels (porins) that selectively admit low molecular weight substrates, including many antibiotics. Improved understanding of the translocation mechanisms of porin substrates could help guide the design of antibiotics capable of achieving high intracellular exposure. Energy barriers to channel entry and exit govern antibiotic fluxes through porins. We have previously reported a hypothesis that the costs of transferring protein solvation to and from bulk medium underlie the barriers to protein‐ligand association and dissociation, respectively, concomitant with the gain and loss of protein‐ligand interactions during those processes. We have now applied this hypothesis to explain the published rates of entry (association) and exit (dissociation) of six antibiotics to/from reconstituted E. coli porin OmpC. WaterMap was used to estimate the total water transfer energies resulting from transient occupation by each antibiotic. Our results suggest that solvation within the porin cavity is highly energetically favorable, and the observed moderately fast entry rates of the antibiotics are consistent with replacement of protein‐water H‐bonds. The observed ultrafast exit kinetics is consistent with the lack of intrachannel solvation sites that convey unfavorable resolvation during antibiotic dissociation. These results are aligned with known general relationships between antibiotic efficacy and physicochemical properties, namely unusually low logP, reflecting an abundance of H‐bond partners. We conclude that antibiotics figuratively “melt” their way through porin solvation at a rate determined by the cost of exchanging protein‐solvent for protein‐antibiotic H‐bonds. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Discovery and structural characterization of peficitinib (ASP015K) as a novel and potent JAK inhibitor

Janus kinases (JAKs) are considered promising targets for the treatment of autoimmune diseases including rheumatoid arthritis (RA) due to their important role in multiple cytokine receptor signaling pathways. Recently, several JAK inhibitors have been developed for the treatment of RA. Here, we describe the identification of the novel orally bioavailable JAK inhibitor 18, peficitinib (also known as ASP015K), which showed moderate selectivity for JAK3 over JAK1, JAK2, and TYK2 in enzyme assays. Chemical modification at the C4-position of lead compound 5 led to a large increase in JAK inhibitory activity and metabolic stability in liver microsomes. Furthermore, we determined the crystal structures of JAK1, JAK2, JAK3, and TYK2 in a complex with peficitinib, and revealed that the 1H-pyrrolo[2,3–b]pyridine-5-carboxamide scaffold of peficitinib forms triple hydrogen bonds with the hinge region. Interestingly, the binding modes of peficitinib in the ATP-binding pockets differed among JAK1, JAK2, JAK3, and TYK2. WaterMap analysis of the crystal structures suggests that unfavorable water molecules are the likely reason for the difference in orientation of the 1H-pyrrolo[2,3-b]pyridine-5-carboxamide scaffold to the hinge region among JAKs.     

In silico identification and design of potent peptide inhibitors against PDZ-3 domain of Postsynaptic Density Protein (PSD-95)

Unique intrinsic properties of peptides like low toxicity, high biological activity, and specificity make them attractive therapeutic agents. PDZ-binding peptide inhibitors have been demonstrated for curing of Alzheimer, Parkinson, Dementia, and other central nervous system ailments. In this article, we report the successful use of an integrated computational protocol to analyze the structural basis of how peptides bind to the shallow groove of the third PDZ domain (PDZ-3) from the postsynaptic density (PSD-95) protein. This protocol employs careful and precise computational techniques for design of new strategy for predicting novel and potent peptides against PDZ protein. We attempted to generate a pharmacophore model using crystal structure of peptide inhibitor bound to the PDZ-3. A highly specific and sensitive generated pharmacophore model was used for screening virtual database generated using different combination of amino acid substitutions as well as decoy peptide database for its sensitivity and specificity. Identified hit peptides were further analyzed by docking studies, and their stability analyzed using solvated molecular dynamics. Quantum Mechanics/Molecular Mechanics (QM/MM) interaction energy and GMX-PBSA scoring schemes were used for ranking of stable peptides. Computational approach applied here generated encouraging results for identifying peptides against PDZ interaction model. The workflow can be further exercised as a virtual screening technique for reducing the search space for candidate target peptides against PDZ domains.