Free energy calculations for theophylline binding to an RNA aptamer: Comparison of MM-PBSA and thermodynamic integration methods

We have applied the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method (J. Srinivasan, T. E. Cheatham, P. Cieplak, P. A. Kollman, and D. A. Case, Journal of the American Chemical Society, 1998, Vol. 120, pp. 9401-9409) to study the interaction of an RNA aptamer with theophylline and its analogs. The MM-PBSA free energy analysis provides a reasonable absolute binding free energy for the RNA aptamer-theophylline complex formation. Energetic analysis reveals that the van der Waals interaction and the nonpolar contribution to solvation provide the basis for the favorable absolute free energy of complex. This trend is similar to other protein-ligand interactions studied previously. The MM-PBSA method also ranks the relative binding energies of five theophylline analogs approximately correctly, but not as well as the more conventional thermodynamic integration calculations, which were carried out to convert theophylline into its analogs. The comparison of MM-PBSA with TI suggests that the MM-PBSA method has some difficulties with the first-solvation-shell energetics.     

Analysis of the binding energies of testosterone, 5alpha-dihydrotestosterone,androstenedione and dehydroepiandrosterone sulfate with an antitestosterone antibody

Molecular dynamics simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) free energy calculations were used to study the binding of testosterone (TES), 5alpha-dihydrotestosterone (5ADHT), androstenedione (AND), and dehydroepiandrosterone sulfate (DHEAS) to the monoclonal antitestosterone antibody 3-C(4)F(5). The relative binding free energy of TES and AND was also calculated with free energy perturbation (FEP) simulations. The antibody 3-C(4)F(5) has a relatively high affinity (3 x 10(8) M(-1)) and on overall good binding profile for testosterone but its cross-reactivity with DHEAS has been the main reason for the failure to use this antibody in clinical immunoassays. The relative binding free energies obtained with the MM-PBSA method were 1.5 kcal/mol for 5ADHT, 3.8 kcal/mol for AND, and 4.3 kcal/mol for DHEAS, as compared to TES. When a water molecule of the ligand binding site, observed in the antibody-TES crystal structure, was explicitly included in MM-PBSA calculations, the relative binding energies were 3.4, 4.9, and 5.4 kcal/mol for 5ADHT, AND, and DHEAS, respectively. The calculated numbers are in correct order but larger than the corresponding experimental energies of 1.3, 1.5, and 2.6 kcal/mol, respectively. The fact that the MM-PBSA method reproduced the relative binding free energies of DHEAS, a steroid having a negatively charged sulfate group, and the neutrally charged TES, 5ADHT, and AND in satisfactory agreement with experiment shows the robustness of the method in predicting relative binding affinities. The 800-ps FEP simulations predicted that the antibody 3-C(4)F(5) binds TES 1.3 kcal/mol tighter than AND. Computational mutagenesis of selected amino acid residues of the ligand binding site revealed that the lower affinities of AND and DHEAS as compared to TES are due to a combined effect of several residues, each contributing a small fraction to the tighter binding of TES. An exception to this is Tyr99H, whose mutation to Ala lowered the binding of DHEAS 0.7 kcal/mol more than the binding of TES. This is probably due to the hydrogen bonding interaction formed between the OH group of Tyr99H and the sulfate group of DHEAS. Computational mutagensis data also showed that the affinity of the steroids to the antitestosterone antibody 3-C(4)F(5) would be enhanced if Trp47H were repositioned so that it would make more extensive contacts with the bound ligands. In addition, the binding of steroids to antitestosterone, antiprogesterone, and antiestradiol antibodies is discussed.     

Investigating specificity of the anti-hypertensive inhibitor WNK463 against With-No-Lysine kinase family isoforms via multiscale simulations

Abstract

The With-No-Lysine (WNK) kinase family plays a significant role in regulating cation-chloride cotransporters, blood pressure and body fluid homeostasis. Mutations in the gene of WNK family, especially in WNK1 and WNK4 are responsible for pseudohypoaldosteronism type II (PHAII), characterized by hypertension. The selective inhibition of WNK1 over other isoforms has created an immense challenge in the design of an ATP competitive inhibitor due to their high conservatism. In this work, we have compared the selectivity of the inhibitor WNK463, which was designed for WNK1 with other WNK family isoforms by comprehensive molecular modeling, docking and molecular dynamics simulations in conjunction with the Molecular Mechanics Poisson-Boltzmann Surface Area method. Our calculations show that the affinity of the inhibitor decreases in the order WNK2?>?WNK1?>?WNK3?>?WNK4, in agreement with the experiment. Our study reveals that the inhibitor is most selective to WNK2 due to decreased polar solvation and configurational entropy compared to other isoforms. Furthermore, our analyses indicated that the nonpolar contribution from the hydrophobic residues and hydrogen bonds in the hinge region gatekeeper residue Met³⁰⁴ of WNK1 and its equivalent residue from other kinases played a critical role in stabilizing the inhibitor against WNK kinases. Residues Lys²³³, Met³⁰⁴, Phe³⁵⁶ and Leu³⁶⁹ of WNK1 were the essential residue differences compared to other isoforms that led to specific interactions thereby forming the basis of molecular binding pattern of binding interactions. Overall, we have identified conserved WNK-inhibitor interactions and elucidated isoform-specific interactions that could be exploited in the design of more potent and selective WNK inhibitors.

Communicated by Ramaswamy H. Sarma     

Molecular dynamics and free energy studies on the Drosophila melanogaster and Leptinotarsa decemlineata ecdysone receptor complexed with agonists: Mechanism for binding and selectivity

The ecdysone receptor is a nuclear hormone receptor that plays a pivotal role in the insect metamorphosis and development. To address the molecular mechanisms of binding and selectivity, the interactions of two typical agonists Ponasterone A and 20-Hydroxyecdysone with Drosophila melanogaster (DME) and Leptinotarsa decemlineata ecdysone (LDE) receptors were investigated by homology modeling, molecular docking, molecular dynamic simulation, and thermodynamic analysis. We discover that 1) the L5-loop, L11-loop, and H12 helix for DME, L7-loop, and L11-loop for LDE are more flexible, which affect the global dynamics of the ligand-binding pocket, thus facilitating the ligand recognition of ecdysone receptor; 2) several key residues (Thr55/Thr37, Phe109/Phe91, Arg95/Arg77, Arg99/Arg81, Phe108/Leu90, and Ala110/Val92) are responsible for the binding of the proteins; 3) the binding-free energy is mainly contributed by the van der Waals forces as well as the electrostatic interactions of ligand and receptor; 4) the computed binding-free energy difference between DME-C1 and LDE-C1 is –4.65 kcal/mol, explains that C1 can form many more interactions with the DME; 5) residues Phe108/Leu90 and Ala110/Val92 have relatively position and orientation difference in the two receptors, accounting most likely for the ligand selectivity of ecdysone receptor from different orders of insects. This study underscores the expectation that different insect pests should be able to discriminate among compounds from different as yet undiscovered compounds, and the results firstly show a structural and functional relay between the agonists and receptors (DME and LDE), which can provide an avenue for the development of target-specific insecticides.

Communicated by Ramaswamy H. Sarma     

Computational investigation of the HIV-1 Rev multimerization using molecular dynamics simulations and binding free energy calculations

The HIV Rev protein mediates the nuclear export of viral mRNA, and is thereby essential for the production of late viral proteins in the replication cycle. Rev forms a large organized multimeric protein-protein complex for proper functioning. Recently, the three-dimensional structures of a Rev dimer and tetramer have been resolved and provide the basis for a thorough structural analysis of the binding interaction. Here, molecular dynamics (MD) and binding free energy calculations were performed to elucidate the forces thriving dimerization and higher order multimerization of the Rev protein. It is found that despite the structural differences between each crystal structure, both display a similar behavior according to our calculations. Our analysis based on a molecular mechanics-generalized Born surface area (MM/GBSA) and a configurational entropy approach demonstrates that the higher order multimerization site is much weaker than the dimerization site. In addition, a quantitative hot spot analysis combined with a mutational analysis reveals the most contributing amino acid residues for protein interactions in agreement with experimental results. Additional residues were found in each interface, which are important for the protein interaction. The investigation of the thermodynamics of the Rev multimerization interactions performed here could be a further step in the development of novel antiretrovirals using structure based drug design. Moreover, the variability of the angle between each Rev monomer as measured during the MD simulations suggests a role of the Rev protein in allowing flexibility of the arginine rich domain (ARM) to accommodate RNA binding.     

Insight into the key active sites of afChiA1 based on molecular dynamics simulations and free energy calculations

In this study, the binding of the enzyme chitinase A1 (afChiA1) from the plant-type Aspergillus fumigatus with four potent inhibitors, allosamidin (ASM), acetazolamide (AZM), 8-chloro-theophylline (CTP) and kinetin (KIT) is investigated by molecular docking, molecular dynamics simulation and binding free energy calculation. The results reveal that the electrostatic interactions play an important role in the stabilisation of the binding of afChiA1 with inhibitors. Based on the binding energy of afChiA1-ligands, the key residues (Gln37 and Trp312) in the active binding pocket of the complex systems are confirmed by molecular mechanics/Poisson–Boltzmann surface area method, and the active inhibitors, ASM and AZM, both could form strong interaction with Gln37 and Trp312, and the non-active ligands, CTP and KIT, could not interact with these two residues, which is consistent with the result of experimental report. Then, it is identified that Gln37 and Trp312 should be one of the important active site residues of afChiA1.     

Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3

The crystal structures of the Fab' fragment of the anti-progesterone monoclonal antibody DB3 and its complexes with steroid haptens have shown that the D-JH junctional residue TrpH100 is a key contributor to binding site interactions with ligands. The indole group of TrpH100 also undergoes a significant conformational change between the bound and unliganded states, effectively opening and closing the combining site pocket. In order to explore the effect of substitutions at this position on steroid recognition, we have carried out mutagenesis on a construct encoding a three-domain single-chain fragment (VH/K) of DB3 expressed in Escherichia coli. TrpH100 was replaced by 13 different amino acids or deleted, and the functional and antigenic properties of the mutated fragments were analyzed. Most substitutions, including small, hydrophobic, hydrophilic, neutral, and negatively charged side chains, were reduced or abolished binding to free progesterone, although binding to progesterone-BSA was partially retained. The reduction in antigen binding was paralleled by alteration of the idiotype associated with the DB3 combining site. In contrast, the replacement of TrpH100 by Arg produced a mutant that retained wild-type antibody affinity and idiotype, but with altered specificity. Significant changes in this mutant included increased relative affinities of 10(4)-fold for progesterone-3-carboxymethyloxime and 10-fold for aetiocholanolone. Our results demonstrate an essential role for the junctional residue H100 in determining steroid-binding specificity and combining site idiotype and show that these properties can be changed by a single amino acid substitution at this position.     

Dynamic control of allosteric antagonism of leukocyte function antigen-1 and intercellular adhesion molecule-1 interaction

Leukocyte function associated antigen-1 (LFA-1) plays a critical role in T cell migration and has been recognized as a therapeutic target for immune disorders. Several classes of small molecule antagonists have been developed to block LFA-1 interaction with intercellular adhesion molecule-1 (ICAM-1). Recent structural studies show that the antagonists bind to an allosteric site in the I-domain of LFA-1. However, it is not yet clear how these small molecules work as antagonists since no significant conformational change is observed in the I-domain-antagonist complex structures. Here we present a computational study suggesting how these allosteric antagonists affect the dynamics of the I-domain. The lowest frequency vibrational mode calculated from an LFA-1 I-domain structure shows large scale "coil-down" motion of the C-terminal alpha7 helix, which may lead to the open form of the I-domain. The presence of an allosteric antagonist greatly reduces this motion of the alpha7 helix as well as other parts of the I-domain. Thus, our study suggests that allosteric antagonists work by eliminating breathing motion that leads to the open conformation of the I-domain.     

Exploring the mechanism how AF9 recognizes and binds H3K9ac by molecular dynamics simulations and free energy calculations

Histone acetylation is a very important regulatory mechanism in gene expression in the chromatin context. A new protein family‐YEATS domains have been found as a novel histone acetylation reader, which could specific recognize the histone lysine acetylation. AF9 is an important one in the YEATS family. Focused on the AF9‐H3K9ac (K9 acetylation) complex (ALY) (PDB code: 4TMP) and a serials of mutants, MUT (the acetyllsine of H3K9ac was mutated to lysine), F59A, G77A, and D103A, we applied molecular dynamics simulation and molecular mechanics Poisson?Boltzmann (MM‐PBSA) free energy calculations to examine the role of AF9 protein in recognition interaction. The simulation results and analysis indicate that some residues of the protein have significant influence on recognition and binding to H3K9ac peptides and hydrophobic surface show the hydrophobic interactions play an important role in the binding. Our work can give important information to understand how the protein AF9 recognizes the peptides H3K9ac. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 779–786, 2016.     

Deciphering the mechanism behind the varied binding activities of COXIBs through Molecular Dynamic Simulations,MM-PBSA binding energy calculations and per-residue energy decomposition studies

COX-2 is a well-known drug target in inflammatory disorders. COX-1/COX-2 selectivity of NSAIDs is crucial in assessing the gastrointestinal side effects associated with COX-1 inhibition. Celecoxib, rofecoxib, and valdecoxib are well-known specific COX-2 inhibiting drugs. Recently, polmacoxib, a COX-2/CA-II dual inhibitor has been approved by the Korean FDA. These COXIBs have similar structure with diverse activity range. Present study focuses on unraveling the mechanism behind the 10-fold difference in the activities of these sulfonamide-containing COXIBs. In order to obtain insights into their binding with COX-2 at molecular level, molecular dynamics simulations studies, and MM-PBSA approaches were employed. Further, per-residue decomposition of these energies led to the identification of crucial amino acids and interactions contributing to the differential binding of COXIBs. The results clearly indicated that Leu338, Ser339, Arg499, Ile503, Phe504, Val509, and Ser516 (Leu352, Ser353, Arg513, Ile517, Phe518, Val523, and Ser530 in PGHS-1 numbering) were imperative in determining the activity of these COXIBs. The binding energies and energy contribution of various residues were similar in all the three simulations. The results suggest that hydrogen bond interaction between the hydroxyl group of Ser516 and five-membered ring of diarylheterocycles augments the affinity in COXIBs. The SAR of the inhibitors studied and the per-residue energy decomposition values suggested the importance of Ser516. Additionally, the positive binding energy obtained with Arg106 explains the binding of COXIBs in hydrophobic channel deep in the COX-2 active site. The findings of the present work would aid in the development of potent COX-2 inhibitors.     

Free energy calculations show that acidic P1 variants undergo large pKa shifts upon binding to trypsin

Serine proteinases and their protein inhibitors belong to one of the most comprehensively studied models of protein-protein interactions. It is well established that the narrow trypsin specificity is caused by the presence of a negatively charged aspartate at the specificity pocket. X-ray crystallography as well as association measurements revealed, surprisingly, that BPTI with glutamatic acid as the primary binding (P1) residue was able to bind to trypsin. Previous free energy calculations showed that there was a substantially unfavorable binding free energy associated with accommodation of ionized P1 Glu at the S1-site of trypsin. In this study, the binding of P1 Glu to trypsin has been systematically investigated in terms of the protonation states of P1 Glu and Asp189, the orientation of Gln192, as well as the possible presence of counterions using the linear interaction energy (LIE) approach and the free energy perturbation (FEP) method. Twenty-four conceivable binding arrangements were evaluated and quantitative agreement with experiments is obtained when the P1 Glu binds in its protonated from. The results suggest that P1 Glu is one of the variants of BPTI that inhibit trypsin strongest at low pH, contrary to the specificity profile of trypsin, suggesting a new regulation mechanism of trypsin-like enzymes.     

Site specific point mutation changes specificity: A molecular modeling study by free energy simulations and enzyme kinetics of the thermodynamics in ribonuclease T1 substrate interactions

We have theoretically and experimentally studied the binding of two different ligands to wild-type ribonuclease T₁ (RNT1) and to a mutant of RNT1 with Glu-46 replaced by Gln. The binding of the natural substrate 3′-GMP has been compared with the binding of a fluorescent probe, 2-aminopurine 3′-monophosphate (2AP), and relative free energies of binding of these ligands to the mutant and the wild-type (wt) enzyme have been calculated by free energy perturbation methods. The free energy perturbations predict that the mutant RNT1-Gln-46 binds 2AP better than 3′GMP, in agreement with experiments on dinucleotides. Four free energy perturbations, forming a closed loop, have been performed to allow the detection of systematic errors in the simulation procedure. Because of the larger number of atoms involved, it was necessary to use a much longer simulation time for the change in the protein, i.e., the perturbation from Glu to Gln, than in the perturbation from 3′-GMP to 2AP. Finally the structure of the binding site is analyzed for understanding differences in catalytic speed and binding strength. © 1993 Wiley-Liss, Inc.     

Application of molecular dynamics and free energy perturbation methods to metalloporphyrin-ligand systems II: CO and dioxygen binding to myoglobin.

The protein contribution to the relative binding affinity of the ligands CO and O2 toward myoglobin (Mb) has been simulated using free energy perturbation calculations. The tautomers of the His E7 residue are different for the oxymyoglobin (MbO2) and carboxymyoglobin (MbCO) systems. This was modeled by performing two-step calculations that mutate the ligand and mutate the His E7 tautomers in separate steps. Differences in hydrogen bonding to the O2 and CO ligands were incorporated into the model. The O2 complex was calculated to be 2-3 kcal/mol more stable than the corresponding CO complex when compared to the same difference in an isolated heme control. This value agrees well with the experimental value of 2.0 kcal/mol. In qualitative agreement with experiments, the Fe-C-O bond is found to be bent (theta = 159.8 degrees) with a small tilt (theta = 6.2 degrees). The contributions made by each of the 29 residues--within the 9.0-A radius of the iron atom--to the free energy difference are separated into van der Waals and electrostatic contributions; the latter contributions are dominant. Aside from the proximal histidine and the heme group, the residues having the largest difference in free energy in mutating MbO2-->MbCO are His E7, Phe CD1, Phe CD4, Val E11, and Thr E10.     

Exploring the binding mechanism of saccharin and sodium saccharin to promoter of human p53 gene by theoretical and experimental methods

Abstract

In the past few decades, extensive discussions have been on the impact of artificial sweeteners on the risk of cancer. The present study aimed to evaluate the interaction of saccharin (SA) and sodium saccharin (SSA) with the promoter of the human p53 gene. The binding ability was assessed using the spectroscopic technique, molecular docking and molecular dynamics (MD) simulation methods. Free energy of binding has been calculated using Molecular Mechanics/Poisson–Boltzmann Surface Area (MM/PBSA) method. Fluorescence spectra of mentioned gene with concentration profiles of SA and SSA were obtained in a physiological condition. A gradual increase without any significant spectral shift in the fluorescence intensity of around 350?nm was evident, indicating the presence of an interaction between both compounds and gene. The docking results showed that both compounds were susceptible to bind to 5′-DG56DG57-3′ nucleotide sequence of gene. Furthermore, the MD simulation demonstrated that the binding positions for SA and SSA were 5′-A1T3T4-3′ and 5′-G44T45-3′ sequences of gene, respectively. The binding of these sweeteners to gene made significant conformational changes to the DNA structure. Hydrogen and hydrophobic interactions are the major forces in complexes stability. Through the groove binding mode, the non-interactive DNA-binding nature of SSA and SA has been demonstrated by the results of spectrofluorometric and molecular modeling. This study could provide valuable insight into the binding mechanism of SA and its salt with p53 gene promoter as macromolecule at the molecular level in atomistic details. This work can contribute to the possibility of the potential hazard of carcinogenicity of this sweetener and to design and apply new and safer artificial sweeteners. Abbreviations SA Saccharin

SSA Sodium Saccharin

Pp53g promoter of human p53 gene

MD Molecular dynamics

RMSD Root-mean-square deviation

RMSF Root-mean-square fluctuation

Rg Radius of Gyration

SASA Solvent-Accessible Surface Area

ADI Acceptable daily intake

MM/PBSA Molecular Mechanics/Poisson–Boltzmann Surface Area

Communicated by Ramaswamy H. Sarma     

Structure modeling, ligand binding, and binding affinity calculation (LR-MM-PBSA) of human heparanase for inhibition and drug design

Human heparanase is an endo-beta-D-glycosidase that cleaves heparan sulphate (HS) chains in the extracellular matrix and basement membrane. It is known that the cleavage of HS by heparanase results in cell invasion and metastasis of cancer. Therefore, heparanase is considered an important target for cancer drug development. The three-dimensional structure of heparanase would be useful in the rational design of inhibitors targeted to the enzyme; however, the three-dimensional structure has not yet been determined. In our effort to design inhibitors, we developed a three-dimensional structure of heparanase using a homology-modeling approach. The homology-built structure is consistent to previous bioinformatics and site-mutation experimental results. The heparanase features a (alpha/beta)(8) TIM-barrel fold with two glutamate residues (Glu225 and Glu343) located in the active-site cleft. This feature supports the putative mechanism of proton donor and nucleophilic sites. Docking simulations yielded 41 complex structures, which indicate that the bound inhibitor could block ligand binding into the catalytic site. A free energy of binding model was established for 25 heparanase inhibitors with a training set of 25 heparanase inhibitors using the linear response MM-PBSA approach (LR-MM-PBSA). The correlation between calculated and experimental activity was 0.79 and the reliability of the model was validated with leave-one-out cross-validation method. Its predictive capability was further validated using a test set of 16 inhibitors similar to the training set of inhibitors. The correlation between the predicted and observed activities is significantly improved by the protein "induced-fit" that accounts for the flexibility of the receptor. These interaction and pharmacophore elements provide a unique insight to the rational design of new ligands targeted to the enzyme.     

Ligand binding and dynamics of the monomeric epidermal growth factor receptor ectodomain

The ectodomain of the human epidermal growth factor receptor (hEGFR) controls input to several cell signalling networks via binding with extracellular growth factors. To gain insight into the dynamics and ligand binding of the ectodomain, the hEGFR monomer was subjected to molecular dynamics simulation. The monomer was found to be substantially more flexible than the ectodomain dimer studied previously. Simulations where the endogeneous ligand EGF binds to either Subdomain I or Subdomain III, or where hEGFR is unbound, show significant differences in dynamics. The molecular mechanics Poisson–Boltzmann surface area method has been used to derive relative free energies of ligand binding, and we find that the ligand is capable of binding either subdomain with a slight preference for III. Alanine‐scanning calculations for the effect of selected ligand mutants on binding reproduce the trends of affinity measurements. Taken together, these results emphasize the possible role of the ectodomain monomer in the initial step of ligand binding, and add details to the static picture obtained from crystal structures. Proteins 2013; 81:1931–1943. © 2013 The Authors. Proteins published by Wiley Periodicals, Inc.     

Free energy simulation to investigate the effect of amino acid sequence environment on the severity of osteogenesis imperfecta by glycine mutations in collagen

Molecular dynamics simulations were carried out to calculate the free energy change difference of two collagen-like peptide models for Gly --> Ser mutations causing two different osteogenesis imperfecta phenotypes. These simulations were performed to investigate the impact of local amino acid sequence environment adjacent to a mutation site on the stability of the collagen. The average free energy differences for a Gly --> Ser mutant relative to a wild type are 3.4 kcal/mol and 8.2 kcal/mol for a nonlethal site and a lethal site, respectively. The free energy change differences of mutant containing two Ser residues relative to the wild type at the nonlethal and lethal mutation sites are 4.6 and 9.8 kcal/mol, respectively. Although electrostatic interactions stabilize mutants containing one or two Ser residues at both mutation sites, van der Waals interactions are of sufficient magnitude to cause a net destabilization. The presence of Gln and Arg near the mutation site, which contain large and polar side chains, provide more destabilization than amino acids containing small and nonpolar side chains.     

Study of the insulin dimerization: binding free energy calculations and per-residue free energy decomposition

A calculation of the binding free energy for the dimerization of insulin has been performed using the molecular mechanics-generalized Born surface area approach. The calculated absolute binding free energy is -11.9 kcal/mol, in approximate agreement with the experimental value of -7.2 kcal/mol. The results show that the dimerization is mainly due to nonpolar interactions. The role of the hydrogen bonds between the 2 monomers appears to give the direction of the interactions. A per-atom decomposition of the binding free energy has been performed to identify the residues contributing most to the self association free energy. Residues B24-B26 are found to make the largest favorable contributions to the dimerization. Other residues situated at the interface between the 2 monomers were found to make favorable but smaller contributions to the dimerization: Tyr B16, Val B12, and Pro B28, and to an even lesser extent, Gly B23. The energy decomposition on a per-residue basis is in agreement with experimental alanine scanning data. The results obtained from a single trajectory (i.e., the dimer trajectory is also used for the monomer analysis) and 2 trajectories (i.e., separate trajectories are used for the monomer and dimer) are similar.