Alchemical Free Energy Perturbation Research Articles

Insights into Membrane Protein-Lipid Interactions from Free Energy Calculations.

Integral membrane proteins are regulated by specific interactions with lipids from the surrounding bilayer. The structures of protein–lipid complexes can be determined through a combination of experimental and computational approaches, but the energetic basis of these interactions is difficult to resolve. Molecular dynamics simulations provide the primary computational technique to estimate the free energies of these interactions. We demonstrate that the energetics of protein–lipid interactions may be reliably and reproducibly calculated using three simulation-based approaches: potential of mean force calculations, alchemical free energy perturbation, and well-tempered metadynamics. We employ these techniques within the framework of a coarse-grained force field and apply them to both bacterial and mammalian membrane protein–lipid systems. We demonstrate good agreement between the different techniques, providing a robust framework for their automated implementation within a pipeline for annotation of newly determined membrane protein structures.

Computing Relative Binding Affinity of Ligands to Receptor: An Effective Hybrid Single-Dual-Topology Free-Energy Perturbation Approach in NAMD.

An effective hybrid single-dual-topology protocol is designed for the calculation of relative binding affinities of small ligands to a receptor. The protocol was developed as an extension of the NAMD molecular dynamics program, which exclusively supports a dual-topology framework for relative alchemical free-energy perturbation (FEP) calculations. In this protocol, the alchemical end states are represented as two separate molecules sharing a common substructure identified through maximum structural mapping. Within the substructure, an atom-to-atom correspondence is established, and each pair of corresponding atoms is holonomically constrained to share identical coordinates at all time throughout the simulation. The forces are projected and combined at each step for propagation. Following this formulation, a set of illustrative calculations of reliable experiment/simulation data, including relative solvation free energies of small molecules and relative binding affinities of drug compounds to proteins, are presented. To enhance sampling of the dual-topology region, the FEP calculations were carried out within a replica-exchange MD scheme supported by the multiple-copy algorithm module of NAMD, with periodically attempted swapping of the thermodynamic coupling parameter λ between neighboring states. The results are consistent with experiments and benchmarks reported in the literature, lending support to the validity of the current protocol. In summary, this hybrid single-dual-topology approach combines the conceptual simplicity of the dual-topology paradigm with the advantageous sampling efficiency of the single-topology approach, making it an ideal strategy for high-throughput in silico drug design.

Upgrading and Validation of the AMBER Force Field for Histidine and Cysteine Zinc(II)-Binding Residues in Sites with Four Protein Ligands.

We developed and validated a novel force field in the context of the AMBER parameterization for the simulation of zinc(II)-binding proteins. The proposed force field assumes nonbonded spherical interactions between the central zinc(II) and the coordinating residues. A crucial innovative aspect of our approach is to account for the polarization effects of the cation by redefining the atomic charges of the coordinating residues and an adjustment of Lennard-Jones parameters of Zn-interacting atoms to reproduce mean distance distributions. The optimal transferable parametrization was obtained by performing accurate quantum mechanical calculations on a training set of high-quality protein structures, encompassing the most common folds of zinc(II) sites. The addressed sites contain a zinc(II) ion tetra-coordinated by histidine and cysteine residues and represent about 70% of all physiologically relevant zinc(II) sites in the Protein Data Bank. Molecular dynamics simulations with explicit solvent, carried out on several zinc(II)-binding proteins not included in the training set, show that our model for zinc(II) sites preserves the tetra-coordination of the metal site with remarkable stability, yielding zinc(II)-X mean distances similar to experimental data. Finally, the model was tested by evaluating the zinc(II)-binding affinities, using the alchemical free energy perturbation approach. The calculated dissociation constants correlate satisfactorily with the experimental counterpart demonstrating the validity and transferability of the proposed parameterization for zinc(II)-binding proteins.

Protein-Ligand Binding Free Energy Calculations with FEP.

Accurate and reliable calculation of protein-ligand binding free energy is of central importance in computational biophysics and structure-based drug design. Among the various methods to calculate protein-ligand binding affinities, alchemical free energy perturbation (FEP) calculations performed by way of explicitly solvated molecular dynamics simulations (FEP/MD) provide a thermodynamically rigorous and complete description of the binding event and should in turn yield highly accurate predictions. Although the original theory of FEP was proposed more than 60years ago, subsequent applications of FEP to compute protein-ligand binding free energies in the context of drug discovery projects over much of that time period was sporadic and generally unsuccessful. This was mainly due to the limited accuracy of the available force fields, inadequate sampling of the protein-ligand conformational space, complexity of simulation set up and analysis, and the large computational resources required to pursue such calculations. Over the past few years, there have been advances in computing power, classical force field accuracy, enhanced sampling algorithms, and simulation setup. This has led to newer FEP implementations such as the FEP+ technology developed by Schrödinger Inc., which has enabled accurate and reliable calculations of protein-ligand binding free energies and positioned free energy calculations to play a guiding role in small-molecule drug discovery. In this chapter, we outline the methodological advances in FEP+, including the OPLS3 force fields, the REST2 (Replica Exchange with Solute Tempering) enhanced sampling, the incorporation of REST2 sampling with conventional FEP (Free Energy Perturbation) through FEP/REST, and the advanced simulation setup and data analysis. The validation of FEP+ method in retrospective studies and the prospective applications in drug discovery projects are also discussed. We then present the recent extension of FEP+ method to handle challenging perturbations, including core-hopping transformations, macrocycle modifications, and reversible covalent inhibitor optimization. The limitations and pitfalls of the current FEP+ methodology and the best practices in real applications are also examined.

A Streamlined, General Approach for Computing Ligand Binding Free Energies and Its Application to GPCR-Bound Cholesterol.

The theory of receptor-ligand binding equilibria has long been well-established in biochemistry, and was primarily constructed to describe dilute aqueous solutions. Accordingly, few computational approaches have been developed for making quantitative predictions of binding probabilities in environments other than dilute isotropic solution. Existing techniques, ranging from simple automated docking procedures to sophisticated thermodynamics-based methods, have been developed with soluble proteins in mind. Biologically and pharmacologically relevant protein-ligand interactions often occur in complex environments, including lamellar phases like membranes and crowded, nondilute solutions. Here, we revisit the theoretical bases of ligand binding equilibria, avoiding overly specific assumptions that are nearly always made when describing receptor-ligand binding. Building on this formalism, we extend the asymptotically exact Alchemical Free Energy Perturbation technique to quantifying occupancies of sites on proteins in a complex bulk, including phase-separated, anisotropic, or nondilute solutions, using a thermodynamically consistent and easily generalized approach that resolves several ambiguities of current frameworks. To incorporate the complex bulk without overcomplicating the overall thermodynamic cycle, we simplify the common approach for ligand restraints by using a single distance-from-bound-configuration (DBC) ligand restraint during AFEP decoupling from protein. DBC restraints should be generalizable to binding modes of most small molecules, even those with strong orientational dependence. We apply this approach to compute the likelihood that membrane cholesterol binds to known crystallographic sites on three GPCRs (β2-adrenergic, 5HT-2B, and μ-opioid) at a range of concentrations. Nonideality of cholesterol in a binary cholesterol:phosphatidylcholine (POPC) bilayer is characterized and consistently incorporated into the interpretation. We find that the three sites exhibit very different affinities for cholesterol: The site on the adrenergic receptor is predicted to be high affinity, with 50% occupancy for 1:109 CHOL:POPC mixtures. The sites on the 5HT-2B and μ-opioid receptor are predicted to be lower affinity, with 50% occupancy for 1:103 CHOL:POPC and 1:102 CHOL:POPC, respectively. These results could not have been predicted from the crystal structures alone.

Roles of conserved tryptophans in trimerization of HIV-1 membrane-proximal external regions: Implications for virucidal design via alchemical free-energy molecular simulations.

The Dual-Action Virolytic Entry Inhibitors, or "DAVEI's," are a class of recombinant fusions of a lectin, a linker polypeptide, and a 15-residue fragment from the membrane-proximal external region (MPER) of HIV-1 gp41. DAVEI's trigger rupture of HIV-1 virions, and the interaction site between DAVEI MPER and HIV-1 lies in the gp41 component of the envelope glycoprotein Env. Here, we explore the hypothesis that DAVEI MPER engages Env gp41 in a mode structurally similar to a crystallographic MPER trimer. We used alchemical free-energy perturbation to assess the thermodynamic roles of each of the four conserved tryptophan residues on each protomer of MPER3 . We found that a W666A mutation had a large positive ΔΔG for all three protomers, while W672A had a large positive ΔΔG for only two of the three protomers, with the other tryptophans remaining unimportant contributors to MPER3 stability. The protomer for which W672 is not important is unique in the placement of its W666 sidechain between the other two protomers. We show that the unique orientation of this W666 sidechain azimuthally rotates its protomer away from the orientation it would have if the trimer were symmetric, resulting in the diminished interaction of this W672 with the rest of MPER3 . Our findings are consistent with our previous experimental study of W-to-A mutants of DAVEI. This suggests that DAVEI MPER may engage HIV-1 Env to form a mixed trimer state in which one DAVEI MPER forms a trimer by displacing a more weakly interacting protomer of the endogenous Env MPER trimer.

Assessing the stability of free-energy perturbation calculations by performing variations in the method

We have calculated relative binding affinities for eight tetrafluorophenyl-triazole-thiogalactoside inhibitors of galectin-3 with the alchemical free-energy perturbation approach. We obtain a mean absolute deviation from experimental estimates of only 2–3 kJ/mol and a correlation coefficient (R2) of 0.5–0.8 for seven relative affinities spanning a range of up to 11 kJ/mol. We also studied the effect of using different methods to calculate the charges of the inhibitor and different sizes of the perturbed group (the atoms that are described by soft-core potentials and are allowed to have differing coordinates). However, the various approaches gave rather similar results and it is not possible to point out one approach as consistently and significantly better than the others. Instead, we suggest that such small and reasonable variations in the computational method can be used to check how stable the calculated results are and to obtain a more accurate estimate of the uncertainty than if performing only one calculation with a single computational setup.

Can System Truncation Speed up Ligand-Binding Calculations with Periodic Free-Energy Simulations?

We have investigated whether alchemical free-energy perturbation calculations of relative binding energies can be sped up by simulating a truncated protein. Previous studies with spherical nonperiodic systems showed that the number of simulated atoms could be reduced by a factor of 26 without affecting the calculated binding free energies by more than 0.5 kJ/mol on average ( Genheden, S.; Ryde, U. J. Chem. Theory Comput. 2012 , 8 , 1449 ), leading to a 63-fold decrease in the time consumption. However, such simulations are rather slow, owing to the need of a large cutoff radius for the nonbonded interactions. Periodic simulations with the electrostatics treated by Ewald summation are much faster. Therefore, we have investigated if a similar speed-up can be obtained also for periodic simulations. Unfortunately, our results show that it is harder to truncate periodic systems and that the truncation errors are larger for these systems. In particular, residues need to be removed from the calculations, which means that atoms have to be restrained to avoid that they move in an unrealistic manner. The results strongly depend on the strength on this restraint. For the binding of seven ligands to dihydrofolate reductase and ten inhibitors of blood-clotting factor Xa, the best results are obtained with a small restraining force constant. However, the truncation errors were still significant (e.g., 1.5-2.9 kJ/mol at a truncation radius of 10 Å). Moreover, the gain in computer time was only modest. On the other hand, if the snapshots are truncated after the MD simulations, the truncation errors are small (below 0.9 kJ/mol even for a truncation radius of 10 Å). This indicates that postprocessing with a more accurate energy function (e.g., with quantum chemistry) on truncated snapshots may be a viable approach.

Methylation-targeted specificity of the DNA binding proteins R.DpnI and MeCP2 studied by molecular dynamics simulations.

DNA methylation plays a major role in organismal development and the regulation of gene expression. Methylation of cytosine bases and the cellular roles of methylated cytosine in eukaryotes are well established, as well as methylation of adenine bases in bacterial genomes. Still lacking, however, is a general mechanistic understanding, in structural and thermodynamic terms, of how proteins recognize methylated DNA. Toward this aim, we present the results of molecular dynamics simulations, alchemical free energy perturbation, and MM-PBSA calculations to explain the specificity of the R.DpnI enzyme from Streptococcus pneumonia in binding to adenine-methylated DNA with both its catalytic and winged-helix domains. We found that adenine-methylated DNA binds more favorably to the catalytic subunit of R.DpnI (-4kcalmol-1) and to the winged-helix domain (-1.6kcalmol-1) than non-methylated DNA. In particular, N6-adenine methylation is found to enthalpically stabilize binding to R.DpnI. In contrast, C5-cytosine methylation entropically favors complexation by the MBD domain of the human MeCP2 protein with almost no contribution of the binding enthalpy.

The Effect of Conformational Flexibility on Binding Free Energy Estimation between Kinases and Their Inhibitors.

Accurate prediction of binding affinities of drug candidates to their targets remains challenging because of protein flexibility in solution. Conformational flexibility of the ATP-binding site in the CDK2 and ERK2 kinases was identified using molecular dynamics simulations. The binding free energy (ΔG) of twenty-four ATP-competitive inhibitors toward these kinases was assessed using an alchemical free energy perturbation method, MP-CAFEE. However, large calculation errors of 2-3 kcal/mol were observed using this method, where the free energy simulation starts from a single equilibrated conformation. Here, we developed a new ΔG computation method, where the starting structure was set to multiconformations to cover flexibility. The calculation accuracy was successfully improved, especially for larger molecular size compounds, leading to reliable prediction of a broader range of drug candidates. The present study demonstrates that conformational flexibility of interactions between a compound and the glycine-rich loop in the kinases is a key factor in ΔG estimation.

A Novel Bifunctional Alkylphenol Anesthetic Allows Characterization of γ-Aminobutyric Acid, Type A (GABAA), Receptor Subunit Binding Selectivity in Synaptosomes

Propofol, an intravenous anesthetic, is a positive modulator of the GABAA receptor, but the mechanistic details, including the relevant binding sites and alternative targets, remain disputed. Here we undertook an in-depth study of alkylphenol-based anesthetic binding to synaptic membranes. We designed, synthesized, and characterized a chemically active alkylphenol anesthetic (2-((prop-2-yn-1-yloxy)methyl)-5-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenol, AziPm-click (1)), for affinity-based protein profiling (ABPP) of propofol-binding proteins in their native state within mouse synaptosomes. The ABPP strategy captured ∼4% of the synaptosomal proteome, including the unbiased capture of five α or β GABAA receptor subunits. Lack of γ2 subunit capture was not due to low abundance. Consistent with this, independent molecular dynamics simulations with alchemical free energy perturbation calculations predicted selective propofol binding to interfacial sites, with higher affinities for α/β than γ-containing interfaces. The simulations indicated hydrogen bonding is a key component leading to propofol-selective binding within GABAA receptor subunit interfaces, with stable hydrogen bonds observed between propofol and α/β cavity residues but not γ cavity residues. We confirmed this by introducing a hydrogen bond-null propofol analogue as a protecting ligand for targeted-ABPP and observed a lack of GABAA receptor subunit protection. This investigation demonstrates striking interfacial GABAA receptor subunit selectivity in the native milieu, suggesting that asymmetric occupancy of heteropentameric ion channels by alkylphenol-based anesthetics is sufficient to induce modulation of activity.

Prediction of cyclohexane-water distribution coefficients for the SAMPL5 data set using molecular dynamics simulations with the OPLS-AA force field.

All-atom molecular dynamics simulations were used to predict water-cyclohexane distribution coefficients [Formula: see text] of a range of small molecules as part of the SAMPL5 blind prediction challenge. Molecules were parameterized with the transferable all-atom OPLS-AA force field, which required the derivation of new parameters for sulfamides and heterocycles and validation of cyclohexane parameters as a solvent. The distribution coefficient was calculated from the solvation free energies of the compound in water and cyclohexane. Absolute solvation free energies were computed by an established protocol using windowed alchemical free energy perturbation with thermodynamic integration. This protocol resulted in an overall root mean square error in [Formula: see text] of almost 4 log units and an overall signed error of -3 compared to experimental data. There was no substantial overall difference in accuracy between simulating in NVT and NPT ensembles. The signed error suggests a systematic error but the experimental [Formula: see text] data on their own are insufficient to uncover the source of this error. Preliminary work suggests that the major source of error lies in the hydration free energy calculations.

Estimating glycosaminoglycan-protein interaction affinity: water dominates the specific antithrombin-heparin interaction.

Glycosaminoglycan (GAG)-protein interactions modulate many important biological processes. Structure-function studies on GAGs may reveal probes and drugs, but their structural complexity and highly acidic nature confound such work. Productivity will increase if we are able to identify tight-binding oligosaccharides in silico. An extension of the CHARMM force field is presented to enable modeling of polysaccharides containing sulfamate functionality, and is used to develop a reliable alchemical free-energy perturbationprotocol that estimates changes in affinity for the prototypical heparin-antithrombin system to within 2.3 kcal/mol using modest simulation times. Inclusion of water is crucial during simulation as solvation energy was equal in magnitude to the sum of all other thermodynamic factors. In summary, we have identified and optimized a reliable method for estimation of GAG-protein binding affinity, and shown that solvation is a crucial component in GAG-protein interactions.

Computational Assessment of Trimethoprim Resistance in Dihydrofolate Reductase

We characterize structural and dynamical changes induced on dihydrofolate reductase(DHFR). We investigate the structural features of the mutant protein that allow it to survive natural selection. Single/double/triple mutants detected via the systematic experimental approach carried out in the morbidostat1 under the selection pressure induced by the antibiotic trimethoprim(TMP), a competitive inhibitor of dihydrofolate, are studied. We investigate the structural features of DHFR that lead to catalytic activity while simultaneously casting out TMP. Binding constant and catalytic activity measurements on different mutants provide conflicting results, implying alternative routes towards conferring drug resistance. We execute extensive molecular dynamics(MD) simulations for the mutants in their folate or TMP bound conformations. We evaluate the experimental findings regarding DHFR drug resistance through structural changes in the enzyme. To understand if the molecules displaying competitive binding to the same region are discriminated by free energy changes affecting binding probabilities, we perform thermodynamic analyses via alchemical free energy perturbation calculations. Another effective factor in DHFR function is the dynamics of the Met20 loop direct contacting folate. These dynamics include an isomerization that is too slow to be directly observed by conventional MD.2 We employ perturbation-response scanning method3 to quantify the interconversion propensity of the conformers. The competing(or synergistic) effects of dynamics and thermodynamics in the mutants are assessed through a combination of these computational approaches. We reveal a trade-off between stability and enzymatic efficiency: Those mutants that have increased affinity for TMP also have larger k_cat values, albeit always below that of the wild-type. Furthermore, mutants disrupt the correlated motions observed in the TMP-bound wild-type enzyme, while that of the DHF-bound remain unaltered.1. Toprak et al., Nature Genet. 44, 101(2012).2. Schnell et al., Annu. Rev. Biophys. Biomol. Struct. 33, 119(2004).3. Atilgan&Atilgan, PLoS Comput. Biol. 5, e1000544(2009).

Relative Affinities of Positive and Negative Modulators of Heteromeric GABA(A) Receptors for Pseudo-Symmetric Intersubunit Binding Sites

Type-A γ-aminobutyric acid receptors (GABAARs) are pentameric ligand-gated ion channels that are ubiquitous to the central nervous system (CNS) and critical for regulating neuronal excitability. These inhibitory receptors, gated by γ-aminobutyric acid (GABA), can be potentiated and also directly activated by certain exogenous and endogenous lipophilic small molecules. Among the various endogenous modulators are derivatives of cholesterol called neurosteroids; neurosteroids that positively modulate GABAARs can function as natural sedatives, anesthetics, anxiolytics and anti-convulsants. The mechanisms underlying modulation of GABAARs by neurosteroids and exogenous modulators like general anesthetics are still poorly understood, in part due to the location of likely quasi-symmetric interaction sites in the transmembrane domain of the receptor, as well as inconclusive results from mutagenesis experiments. In particular, the extent to which binding modes of positively and negatively modulating neurosteroids overlap is unknown. Here we use molecular dynamics simulations and the thermodynamically rigorous alchemical free energy perturbation technique to rank pseudo-symmetric intersubunit binding modes by affinity for the positively modulating neurosteroid allopregnanolone, the negatively modulating neurosteroid pregnenolone sulfate, and a novel analog with complex dose-response: triiodothyronine (thyroid hormone). Differential rankings of intersubunit sites for positive and negative modulating neurosteroids, triiodothyronine, and the general anesthetics propofol and sevoflurane, are interpreted in the context of their distinctive functional effects.

Dissecting Functional Correlates of a Double Mutation Enhancing GltPh Transport Efficiency using Alchemical Free Energy Calculations

Excitatory amino acid transporters (EAATs) mediate glutamate re-uptake in neurons and glial cells, and are involved in a number of neurodegenerative diseases. An archaeal analog, GltPh, in which two key residues (R276 and M395) are replaced by their counterpart in human EAATs exhibits lower substrate affinity, increased frequency of transport domain motions and increased rate of substrate uptake compared to the wild type (WT) GltPh. Crystallographic analyses showed that transport domains of this mutant can adopt “unlocked” inward-facing conformations with dramatically more opened interfaces than previously reported in WT GltPh. These observations provide a structural rationale for key functional distinctions between the WT GltPh and the human EAATs that exhibit much more rapid substrate transport. However, the mechanistic relation between the mutations and the structural rearrangements remains unknown. The allosteric mechanisms by which the mutations act to decrease substrate affinity and destabilize the “locked” state are of great interest for attempts to develop therapeutic interventions utilizing improved transport efficiency. To investigate the mechanism we use extensive molecular dynamics simulations and the alchemical free energy perturbation methodology to evaluate free energy differences between WT and the R276S/M395R mutant construct for each of the known conformational states of GltPh, both in their apo and ligand-bound form. Closing the thermodynamic cycles connecting WT and mutant in relevant pairs of conformations allows us to dissect and quantify the contributions of the mutation to different mechanisms that potentially influence transport efficiency: transport domain unlocking, translocation from outward-facing to inward-facing, as well as intracellular and extracellular ligand affinities.

Structural Determinants for the Binding of Morphinan Agonists to the μ-Opioid Receptor.

Atomistic descriptions of the μ-opioid receptor (μOR) noncovalently binding with two of its prototypical morphinan agonists, morphine (MOP) and hydromorphone (HMP), are investigated using molecular dynamics (MD) simulations. Subtle differences between the binding modes and hydration properties of MOP and HMP emerge from the calculations. Alchemical free energy perturbation calculations show qualitative agreement with in vitro experiments performed in this work: indeed, the binding free energy difference between MOP and HMP computed by forward and backward alchemical transformation is 1.2±1.1 and 0.8±0.8 kcal/mol, respectively, to be compared with 0.4±0.3 kcal/mol from experiment. Comparison with an MD simulation of μOR covalently bound with the antagonist β-funaltrexamine hints to agonist-induced conformational changes associated with an early event of the receptor’s activation: a shift of the transmembrane helix 6 relative to the transmembrane helix 3 and a consequent loss of the key R165-T279 interhelical hydrogen bond. This finding is consistent with a previous proposal suggesting that the R165-T279 hydrogen bond between these two helices indicates an inactive receptor conformation.

How to deal with multiple binding poses in alchemical relative protein-ligand binding free energy calculations.

Recent advances in improved force fields and sampling methods have made it possible for the accurate calculation of protein–ligand binding free energies. Alchemical free energy perturbation (FEP) using an explicit solvent model is one of the most rigorous methods to calculate relative binding free energies. However, for cases where there are high energy barriers separating the relevant conformations that are important for ligand binding, the calculated free energy may depend on the initial conformation used in the simulation due to the lack of complete sampling of all the important regions in phase space. This is particularly true for ligands with multiple possible binding modes separated by high energy barriers, making it difficult to sample all relevant binding modes even with modern enhanced sampling methods. In this paper, we apply a previously developed method that provides a corrected binding free energy for ligands with multiple binding modes by combining the free energy results from multiple alchemical FEP calculations starting from all enumerated poses, and the results are compared with Glide docking and MM-GBSA calculations. From these calculations, the dominant ligand binding mode can also be predicted. We apply this method to a series of ligands that bind to c-Jun N-terminal kinase-1 (JNK1) and obtain improved free energy results. The dominant ligand binding modes predicted by this method agree with the available crystallography, while both Glide docking and MM-GBSA calculations incorrectly predict the binding modes for some ligands. The method also helps separate the force field error from the ligand sampling error, such that deviations in the predicted binding free energy from the experimental values likely indicate possible inaccuracies in the force field. An error in the force field for a subset of the ligands studied was identified using this method, and improved free energy results were obtained by correcting the partial charges assigned to the ligands. This improved the root-mean-square error (RMSE) for the predicted binding free energy from 1.9 kcal/mol with the original partial charges to 1.3 kcal/mol with the corrected partial charges.

Affinity Calculations for Lipophilic Modulators Binding to Isolated Sites on GABA(A) Receptors

The γ-amino butyric acid type A (GABA(A)) receptor is an ionotropic receptor critical for inhibitory signaling in the central nervous system. GABA(A) receptors are positively modulated by numerous general anesthetics and likely contribute to the effects of general anesthesia. Several site-directed mutagenesis and photoaffinity labeling experiments have suggested numerous potential binding sites for general anesthetics, as well as other modulators of GABA(A) receptors including neurosteroids and thyroid hormone. The absolute affinities of the modulator for each site are unknown, however, making it challenging to determine which of the many sites is occupied at clinical concentrations. Measurement of modulator affinity for individual sites is challenging experimentally, while isolation of individual sites in computational methods is relatively straightforward. Here we use the Alchemical Free Energy Perturbation (AFEP) method combined with molecular dynamics simulations to calculate absolute binding affinities of propofol and sevoflurane for pore and intersubunit sites on a homology model of an α1β3γ2 GABA(A) receptor, after initial coordinates were determined with automated docking. We calculate significant differences in general anesthetic affinity for pore and intersubunit sites at various interfaces, consistent with a complex dose-response curve in which low affinity sites are associated with inhibition and high affinity sites are associated with potentiation.

How MeCP2 and R.DpnI Proteins Recognize Methylated DNA

DNA methylation plays a major role in organismal development and the regulation of gene expression. Methylation of cytosine bases and its cellular roles in eukaryotes are well established, as well as methylation of adenine bases in bacterial genomes. Here, we present results from molecular dynamics simulations, alchemical free energy perturbation, and MM-PBSA calculations to explain the specificity of the R.DpnI enzyme for binding to adenine-methylated DNA in both its catalytic and winged-helix domains. We find that adenine-methylated DNA binds more favorably to the catalytic subunit of R.DpnI (−4 kcal/mol) and to the winged-helix domain (−1.6 kcal/mol) than unmethylated DNA.In particular, N6-adenine methylation is found to enthalpically stabilize binding to R.DpnI. In contrast, C5-cytosine methylation stabilizes binding to the MBD domain of the MeCP2 entropically with almost no difference in binding enthalpy.