Free enthalpies of replacing water molecules in protein binding pockets

Water molecules in the binding pocket of a protein and their role in ligand binding have increasingly raised interest in recent years. Displacement of such water molecules by ligand atoms can be either favourable or unfavourable for ligand binding depending on the change in free enthalpy. In this study, we investigate the displacement of water molecules by an apolar probe in the binding pocket of two proteins, cyclin-dependent kinase 2 and tRNA-guanine transglycosylase, using the method of enveloping distribution sampling (EDS) to obtain free enthalpy differences. In both cases, a ligand core is placed inside the respective pocket and the remaining water molecules are converted to apolar probes, both individually and in pairs. The free enthalpy difference between a water molecule and a CH₃ group at the same location in the pocket in comparison to their presence in bulk solution calculated from EDS molecular dynamics simulations corresponds to the binding free enthalpy of CH₃ at this location. From the free enthalpy difference and the enthalpy difference, the entropic contribution of the displacement can be obtained too. The overlay of the resulting occupancy volumes of the water molecules with crystal structures of analogous ligands shows qualitative correlation between experimentally measured inhibition constants and the calculated free enthalpy differences. Thus, such an EDS analysis of the water molecules in the binding pocket may give valuable insight for potency optimization in drug design.     

Coarse‐grained molecular dynamics simulations of protein–ligand binding

Coarse‐grained molecular dynamics (CGMD) simulations with the MARTINI force field were performed to reproduce the protein–ligand binding processes. We chose two protein–ligand systems, the levansucrase–sugar (glucose or sucrose), and LinB–1,2‐dichloroethane systems, as target systems that differ in terms of the size and shape of the ligand‐binding pocket and the physicochemical properties of the pocket and the ligand. Spatial distributions of the Coarse‐grained (CG) ligand molecules revealed potential ligand‐binding sites on the protein surfaces other than the real ligand‐binding sites. The ligands bound most strongly to the real ligand‐binding sites. The binding and unbinding rate constants obtained from the CGMD simulation of the levansucrase–sucrose system were approximately 10 times greater than the experimental values; this is mainly due to faster diffusion of the CG ligand in the CG water model. We could obtain dissociation constants close to the experimental values for both systems. Analysis of the ligand fluxes demonstrated that the CG ligand molecules entered the ligand‐binding pockets through specific pathways. The ligands tended to move through grooves on the protein surface. Thus, the CGMD simulations produced reasonable results for the two different systems overall and are useful for studying the protein–ligand binding processes. © 2014 Wiley Periodicals, Inc.     

Molecular dynamics simulations of ligand-induced backbone conformational changes in the binding site of the periplasmic lysine-, arginine-, ornithine-binding protein

The periplasmic lysine-, arginine-, ornithine-binding protein (LAOBP) traps its ligands by a large hinge bending movement between two globular domains. The overall geometry of the binding site remains largely unchanged between the open (unliganded) and closed (liganded) forms, with only a small number of residues exhibiting limited movement of their side chains. However, in the case of the ornithine-bound structure, the backbone peptide bond between Asp11 and Thr12 undergoes a large rotation. Molecular dynamics simulations have been used to investigate the origin and mechanism of this backbone movement. Simulations allowing flexibility of a limited region and of the whole binding site, with and without bound ligands, suggest that this conformational change is induced by the binding of ornithine, leading to the stabilisation of an energetically favourable alternative conformation.     

Computation of binding free energy with molecular dynamics and grand canonical Monte Carlo simulations

The binding of a ligand to a receptor is often associated with the displacement of a number of bound water molecules. When the binding site is exposed to the bulk region, this process may be sampled adequately by standard unbiased molecular dynamics trajectories. However, when the binding site is deeply buried and the exchange of water molecules with the bulk region may be difficult to sample, the convergence and accuracy in free energy perturbation (FEP) calculations can be severely compromised. These problems are further compounded when a reduced system including only the region surrounding the binding site is simulated. To address these issues, we couple molecular dynamics (MD) with grand canonical Monte Carlo (GCMC) simulations to allow the number of water to fluctuate during an alchemical FEP calculation. The atoms in a spherical inner region around the binding pocket are treated explicitly while the influence of the outer region is approximated using the generalized solvent boundary potential (GSBP). At each step during thermodynamic integration, the number of water in the inner region is equilibrated with GCMC and energy data generated with MD is collected. Free energy calculations on camphor binding to a deeply buried pocket in cytochrome P450cam, which causes about seven water molecules to be expelled, are used to test the method. It concluded that solvation free energy calculations with the GCMC/MD method can greatly improve the accuracy of the computed binding free energy compared to simulations with fixed number of water.     

WATsite: Hydration site prediction program with PyMOL interface

Water molecules that mediate protein–ligand interactions or are released from the binding site on ligand binding can contribute both enthalpically and entropically to the free energy of ligand binding. To elucidate the thermodynamic profile of individual water molecules and their potential contribution to ligand binding, a hydration site analysis program WATsite was developed together with an easy‐to‐use graphical user interface based on PyMOL. WATsite identifies hydration sites from a molecular dynamics simulation trajectory with explicit water molecules. The free energy profile of each hydration site is estimated by computing the enthalpy and entropy of the water molecule occupying a hydration site throughout the simulation. The results of the hydration site analysis can be displayed in PyMOL. A key feature of WATsite is that it is able to estimate the protein desolvation free energy for any user specified ligand. The WATsite program and its PyMOL plugin are available free of charge from http://people.pnhs.purdue.edu/~mlill/software . © 2014 Wiley Periodicals, Inc.     

Hydration structure of human lysozyme investigated by molecular dynamics simulation and cryogenic X-ray crystal structure analyses: on the correlation between crystal water sites,solvent density,and solvent dipole

The hydration structure of human lysozyme was studied with cryogenic X-ray diffraction experiment and molecular dynamics simulations. The crystal structure analysis at a resolution of 1.4 A provided 405 crystal water molecules around the enzyme. In the simulations at 300 K, the crystal structure was immersed in explicit water molecules. We examined correlations between crystal water sites and two physical quantities calculated from the 1-ns simulation trajectories: the solvent density reflecting the time-averaged distribution of water molecules, and the solvent dipole measuring the orientational ordering of water molecules around the enzyme. The local high solvent density sites were consistent with the crystal water sites, and better correlation was observed around surface residues with smaller conformational fluctuations during the simulations. Solvent dipoles around those sites exhibited coherent and persistent ordering, indicating that the hydration water molecules at the crystal water sites were highly oriented through the interactions with hydrophilic residues. Those water molecules restrained the orientational motions of adjoining water molecules and induced a solvent dipole field, which was persistent during the simulations around the enzyme. The coherent ordering was particularly prominent in and around the active site cleft of the enzyme. Because the ordering was significant up to the third to fourth solvent layer region from the enzyme surface, the coherently ordered solvent dipoles likely contributed to the molecular recognition of the enzyme in a long-distance range. The present work may provide a new approach combining computational and the experimental studies to understand protein hydration.     

Effect of ligand binding on the intraminimum dynamics of proteins

Effects of ligand binding on protein dynamics are studied via molecular dynamics (MD) simulations on two different enzymes, dihydrofolate reductase (DHFR) and triosephosphate isomerase (TIM), in their unliganded (free) and liganded states. Domain motions in MD trajectories are analyzed by collectivities and rotation angles along the principal components (PCs). DHFR in the free state has well‐defined domain rotations, whereas rotations are slightly damped in the binary complex with nicotinamide adenine dinucleotide phosphate (NADPH), and remarkably distorted in the presence of NADP⁺, showing that NADP⁺ is solely responsible for the loss of correlation of the domains in DHFR. Although mean square fluctuations of MD simulations in the same PC subspaces are similar for different ligation states, linear stochastic time series models show that backbone flexibility along the first five PCs is decreased upon NADPH and NADP⁺ binding in subpicosecond scale. This shows that mobility of the protein along the PCs is closely related with intraminimum dynamics, and alterations in ligation states may change the intraminimum dynamics significantly. Low vibrational frequencies of the alpha‐carbon atoms of DHFR are determined from the time series models of a larger number of low indexed PCs, and it is found that number of modes in the lowest frequencies is reduced upon ligand binding. A similar result is obtained for TIM in the unliganded and dihydroxyacetone phosphate bound states. We suggest that stochastic time series modeling is a promising method to be used in determining subtle perturbations in protein dynamics. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2011     

How does a drug molecule find its target binding site?

Although the thermodynamic principles that control the binding of drug molecules to their protein targets are well understood, detailed experimental characterization of the process by which such binding occurs has proven challenging. We conducted relatively long, unguided molecular dynamics simulations in which a ligand (the cancer drug dasatinib or the kinase inhibitor PP1) was initially placed at a random location within a box that also contained a protein (Src kinase) to which that ligand was known to bind. In several of these simulations, the ligand correctly identified its target binding site, forming a complex virtually identical to the crystallographically determined bound structure. The simulated trajectories provide a continuous, atomic-level view of the entire binding process, revealing persistent and noteworthy intermediate conformations and shedding light on the role of water molecules. The technique we employed, which does not assume any prior knowledge of the binding site's location, may prove particularly useful in the development of allosteric inhibitors that target previously undiscovered binding sites.     

Rational design of peptide ligand for affinity chromatography of tissue-type plasminogen activator by the combination of docking and molecular dynamics simulations

Combined docking and molecular dynamics (MD) simulations are carried out for the rational design of affinity peptide ligand of tissue-type plasminogen activator (t-PA). Ten amino acids that have high affinity to three different regions of t-PA are identified by the amino acids location method on the basis of candidate pocket structure of t-PA. Then, 14 tetrapeptides are built and docked into the candidate pocket of t-PA. The absolute value of the D(score) calculated from the docking simulation is used to assess the affinity of a peptide for t-PA. Consequently, six tetrapeptides that have high D(score) values are selected and linked to a spacer arm of [NH(CH(2))(6)NH(2)] that is present on EAH Sepharose gel. The linked compounds are further evaluated by docking into the candidate pocket of t-PA. As a result, the tetrapeptide QDES with the highest D(score) value is selected. Molecular surface analysis with the MOLCAD program reveals that electrostatic interactions and hydrogen bonds (H-bonds) contribute to the affinity interactions between the tetrapeptide and t-PA. MD simulations indicate that QDES-t-PA complex keeps stable, and the distances between the carboxyl groups of Asp189, Gln192 and Asp194 and the charged amino group of glutamine change little. Moreover, all the nine H-bonds found in the docking simulation are confirmed by the MD simulations. It is also found that three water molecules act as bridges between the ligand and the protein pocket by hydrogen bonding. Finally, high binding affinity and specificity of the peptide ligand are confirmed by the purification of t-PA from crude porcine heart extract using the immobilized-ligand column for affinity chromatography.     

Water molecules inside protein structure affect binding of monosaccharides with HIV‐1 antibody 2G12

Water molecules inside biomolecules constitute integral parts of their structure and participate in the functions of the proteins. Some of the X‐ray crystallographic data are insufficient for analyzing a series of ligand–protein complexes in the same condition. We theoretically investigated antibody binding abilities of saccharide ligands and the effects of the inner water molecules of ligand–antibody complexes. Classical molecular dynamics and quantum chemical simulations using a model with possible water molecules inside the protein were performed with saccharide ligands and Human Immunodeficiency Virus 1 neutralizing antibody 2G12 complexes to estimate how inner water molecules of the protein affect the dynamics of the complexes as well as the ligand–antibody interaction. Our results indicate the fact that d ‐fructose's strong affinity to the antibody was partly due to the good retentiveness of solvent water molecules of the ligand and its stability of the ligand's conformation and relative position in the active site. © 2016 Wiley Periodicals, Inc.     

Open science grid study of the coupling between conformation and water content in the interior of a protein

Computational grids are a promising resource for modeling complex biochemical processes such as protein folding, penetration of gases or water into proteins, or protein structural rearrangements coupled to ligand binding. We have enabled the molecular dynamics program CHARMM to run on the Open Science Grid. The implementation is general, flexible, easily modifiable for use with other molecular dynamics programs and other grids and automated in terms of job submission, monitoring, and resubmission. The usefulness of grid computing was demonstrated through the study of hydration of the Glu-66 side chain in the interior of protein staphylococcal nuclease. Multiple simulations started with and without two internal water molecules shown crystallographically to be associated with the side chain of Glu-66 yielded two distinct populations of rotameric states of Glu-66 that differed by as much as 20%. This illustrates how internal water molecules can bias protein conformations. Furthermore, there appeared to be a temporal correlation between dehydration of the side chain and conformational transitions of Glu-66. This example demonstrated how difficult it is to get convergence even in the relatively simple case of a side chain oscillating between two conformations. With grid computing, we also benchmarked the self-guided Langevin dynamics method against the Langevin dynamics method traditionally used for temperature control in molecular dynamics simulations and showed that the two methods yield comparable results.     

Equatorial and apical solvent shells of the UO2 2+ ion

First principles molecular dynamics simulations of the hydration shells surrounding UO(2)(2+) ions are reported for temperatures near 300 K. Most of the simulations were done with 64 solvating water molecules (22 ps). Simulations with 122 water molecules (9 ps) were also carried out. The hydration structure predicted from the simulations was found to agree with very well-known results from x-ray data. The average U=O bond length was found to be 1.77 A. The first hydration shell contained five trigonally coordinated water molecules that were equatorially oriented about the O-U-O axis with the hydrogen atoms oriented away from the uranium atom. The five waters in the first shell were located at an average distance of 2.44 A (2.46 A, 122 water simulation). The second hydration shell was composed of distinct equatorial and apical regions resulting in a peak in the U-O radial distribution function at 4.59 A. The equatorial second shell contained ten water molecules hydrogen bonded to the five first shell molecules. Above and below the UO(2)(2+) ion, the water molecules were found to be significantly less structured. In these apical regions, water molecules were found to sporadically hydrogen bond to the oxygen atoms of the UO(2)(2+), oriented in such a way as to have their protons pointed toward the cation. While the number of apical waters varied greatly, an average of five to six waters was found in this region. Many water transfers into and out of the equatorial and apical second solvation shells were observed to occur on a picosecond time scale via dissociative mechanisms. Beyond these shells, the bonding pattern substantially returned to the tetrahedral structure of bulk water.     

Structure and dynamics of the hydration shells of the Al3+ ion

First principles simulations of the hydration shells surrounding Al3+ ions are reported for temperatures near 300 degrees C. The predicted six water molecules in the octahedral first hydration shell were found to be trigonally coordinated via hydrogen bonds to 12 s shell water molecules in agreement with the putative structure used to analyze the x-ray data, but in disagreement with the results reported from conventional molecular dynamics using two-and three-body potentials. Bond lengths and angles of the water molecules in the first and second hydration shells and the average radii of these shells also agreed very well with the results of the x-ray analysis. Water transfers into and out of the second solvation shell were observed to occur on a picosecond time scale via a dissociative mechanism. Beyond the second shell the bonding pattern substantially returned to the tetrahedral structure of bulk water. Most of the simulations were done with 64 solvating water molecules (20 ps). Limited simulations with 128 water molecules (7 ps) were also carried out. Results agreed as to the general structure of the solvation region and were essentially the same for the first and second shell. However, there were differences in hydrogen bonding and Al-O radial distribution function in the region just beyond the second shell. At the end of the second shell a nearly zero minimum in the Al-O radial distribution was found for the 128 water system. This minimum is less pronounced minimum found for the 64 water system, which may indicate that sizes larger than 64 may be required to reliably predict behavior in this region.     

Ultrafast hydration dynamics in melittin folding and aggregation: helix formation and tetramer self-assembly

Melittin, an amphipathic peptide from honeybee venom, consists of 26 amino acid residues and adopts different conformations from a random coil, to an alpha-helix, and to a self-assembled tetramer under certain aqueous environments. We report here our systematic studies of the hydration dynamics in these conformations using single intrinsic tryptophan (W19) as a molecular probe. With femtosecond resolution, we observed the solvation dynamics occurring in 0.62 and 14.7 ps in a random-coiled primary structure. The former represents bulklike water motion, and the latter reflects surface-type hydration dynamics of proteins. As a comparison, a model tripeptide (KWK) was also studied. At a membrane-water interface, melittin folds into a secondary alpha-helical structure, and the interfacial water motion was found to take as long as 114 ps, indicating a well-ordered water structure along the membrane surface. In high-salt aqueous solution, the dielectric screening and ionic solvation promote the hydrophobic core collapse in melittin aggregation and facilitate the tetramer formation. This self-assembled tertiary structure is also stabilized by the strong hydrophilic interactions of charged C-terminal residues and associated ions with water molecules in the two assembled regions. The hydration dynamics was observed to occur in 87 ps, significantly slower than typical water relaxation at protein surfaces but similar to water motion at membrane interfaces. Thus, the observed time scale of approximately 100 ps probably implies appropriate water mobility for mediating the formation of high-order structures of melittin in an alpha-helix and a self-assembled tetramer. These results elucidate the critical role of hydration dynamics in peptide conformational transitions and protein structural stability and integrity.     

Toward quantitative simulations of carbon monoxide escape pathways in myoglobin

Straightforward molecular dynamics trajectories have been computed to explore the diffusion of carbon monoxide through myoglobin. The classical equations of motion were integrated for 2 ns and the resulting pathways analyzed. Two types of runs were examined. Type i: Myoglobin and a ligand embedded in a periodic box with 9996 water molecules; the water molecules are rigid but the bonds of the protein are flexible. Type ii: Myoglobin with a solvation shell (153 water molecules) in which all bond lengths are fixed. In trajectories of type i, the diffusing ligand visits a significant part of the protein matrix and was not constrained to the proximity of the heme pocket before escaping. The maximum time of the trajectories was 2 ns. It was shorter if the ligand escaped earlier. Two ligands (from a total of 88) escape to the solvent from nonclassical gates (non-E-helix gates). In trajectories of type ii, the overall fluctuations of the protein are smaller and the ligand explores significantly smaller internal space. The escape rate from type ii trajectories (11 of 400) is comparable to type i and is not dramatically different from experiment (1 of 100). Interestingly, the two simulations with comparable rates sampled different pathways. In trajectories of type ii, we observe escapes from the classical gate (His 64) and from the Xe4 cavity. Further studies (that are underway) are required to define the escape pathways and the overall rate.     

Van der Waals interactions dominate ligand-protein association in a protein binding site occluded from solvent water

In the present study we examine the enthalpy of binding of 2-methoxy-3-isobutylpyrazine (IBMP) to the mouse major urinary protein (MUP), using a combination of isothermal titration calorimetry (ITC), NMR, X-ray crystallography, all-atom molecular dynamics simulations, and site-directed mutagenesis. Global thermodynamics data derived from ITC indicate that binding is driven by favorable enthalpic contributions, rather than a classical entropy-driven signature that might be expected given that the binding pocket of MUP-1 is very hydrophobic. The only ligand-protein hydrogen bond is formed between the side-chain hydroxyl of Tyr120 and the ring nitrogen of the ligand in the wild-type protein. ITC measurements on the binding of IBMP to the Y120F mutant demonstrate a reduced enthalpy of binding, but nonetheless binding is still enthalpy dominated. A combination of solvent isotopic substitution ITC measurements and all-atom molecular dynamics simulations with explicit inclusion of solvent water suggests that solvation is not a major contributor to the overall binding enthalpy. Moreover, hydrogen/deuterium exchange measurements suggest that there is no significant contribution to the enthalpy of binding derived from "tightening" of the protein structure. Data are consistent with binding thermodynamics dominated by favorable dispersion interactions, arising from the inequality of solvent-solute dispersion interactions before complexation versus solute-solute dispersion interactions after complexation, by virtue of poor solvation of the binding pocket.     

Hydration dynamics and time scales of coupled water-protein fluctuations

We report experimental and theoretical studies on water and protein dynamics following photoexcitation of apomyoglobin. Using site-directed mutation and with femtosecond resolution, we experimentally observed relaxation dynamics with a biphasic distribution of time scales, 5 and 87 ps, around the site Trp7. Theoretical studies using both linear response and direct nonequilibrium molecular dynamics (MD) calculations reproduced the biphasic behavior. Further constrained MD simulations with either frozen protein or frozen water revealed the molecular mechanism of slow hydration processes and elucidated the role of protein fluctuations. Observation of slow water dynamics in MD simulations requires protein flexibility, regardless of whether the slow Stokes shift component results from the water or protein contribution. The initial dynamics in a few picoseconds represents fast local motions such as reorientations and translations of hydrating water molecules, followed by slow relaxation involving strongly coupled water-protein motions. We observed a transition from one isomeric protein configuration to another after 10 ns during our 30 ns ground-state simulation. For one isomer, the surface hydration energy dominates the slow component of the total relaxation energy. For the other isomer, the slow component is dominated by protein interactions with the chromophore. In both cases, coupled water-protein motion is shown to be necessary for observation of the slow dynamics. Such biologically important water-protein motions occur on tens of picoseconds. One significant discrepancy exists between theory and experiment, the large inertial relaxation predicted by simulations but clearly absent in experiment. Further improvements required in the theoretical model are discussed.