Insights into protein-protein binding by binding free energy calculation and free energy decomposition for the Ras-Raf and Ras-RalGDS complexes

Absolute binding free energy calculations and free energy decompositions are presented for the protein-protein complexes H-Ras/C-Raf1 and H-Ras/RalGDS. Ras is a central switch in the regulation of cell proliferation and differentiation. In our study, we investigate the capability of the molecular mechanics (MM)-generalized Born surface area (GBSA) approach to estimate absolute binding free energies for the protein-protein complexes. Averaging gas-phase energies, solvation free energies, and entropic contributions over snapshots extracted from trajectories of the unbound proteins and the complexes, calculated binding free energies (Ras-Raf: -15.0(+/-6.3)kcal mol(-1); Ras-RalGDS: -19.5(+/-5.9)kcal mol(-1)) are in fair agreement with experimentally determined values (-9.6 kcal mol(-1); -8.4 kcal mol(-1)), if appropriate ionic strength is taken into account. Structural determinants of the binding affinity of Ras-Raf and Ras-RalGDS are identified by means of free energy decomposition. For the first time, computationally inexpensive generalized Born (GB) calculations are applied in this context to partition solvation free energies along with gas-phase energies between residues of both binding partners. For selected residues, in addition, entropic contributions are estimated by classical statistical mechanics. Comparison of the decomposition results with experimentally determined binding free energy differences for alanine mutants of interface residues yielded correlations with r(2)=0.55 and 0.46 for Ras-Raf and Ras-RalGDS, respectively. Extension of the decomposition reveals residues as far apart as 25A from the binding epitope that can contribute significantly to binding free energy. These "hotspots" are found to show large atomic fluctuations in the unbound proteins, indicating that they reside in structurally less stable regions. Furthermore, hotspot residues experience a significantly larger-than-average decrease in local fluctuations upon complex formation. Finally, by calculating a pair-wise decomposition of interactions, interaction pathways originating in the binding epitope of Raf are found that protrude through the protein structure towards the loop L1. This explains the finding of a conformational change in this region upon complex formation with Ras, and it may trigger a larger structural change in Raf, which is considered to be necessary for activation of the effector by Ras.     

Theoretical study of the ligand-CYP2B4 complexes: effect of structure on binding free energies and heme spin state

The molecular origins of temperature-dependent ligand-binding affinities and ligand-induced heme spin state conversion have been investigated using free energy analysis and DFT calculations for substrates and inhibitors of cytochrome P450 2B4 (CYP2B4), employing models of CYP2B4 based on CYP2C5(3LVdH)/CYP2C9 crystal structures, and the results compared with experiment. DFT calculations indicate that large heme-ligand interactions (ca. -15 kcal/mol) are required for inducing a high to low spin heme transition, which is correlated with large molecular electrostatic potentials (approximately -45 kcal/mol) at the ligand heteroatom. While type II ligands often contain oxygen and nitrogen heteroatoms that ligate heme iron, DFT results indicate that BP and MF heme complexes, with weak substrate-heme interactions (ca. -2 kcal/mol), and modest MEPS minima (>-35 kcal/mol) are high spin. In contrast, heme complexes of the CYP2B4 inhibitor, 4PI, the product of benzphetamine metabolism, DMBP, and water are low spin, have substantial heme-ligand interaction energies (<-15 kcal/mol) and deep MEPS minima (<-45 kcal/mol) near their heteroatoms. MMPBSA analysis of MD trajectories were made to estimate binding free energies of these ligands at the heme binding site of CYP2B4. In order to initially assess the realism of this approach, the binding free energy of 4PI inhibitor was computed and found to be a reasonable agreement with experiment: -7.7 kcal/mol [-7.2 kcal/mol (experiment)]. BP was determined to be a good substrate [-6.3 kcal/mol (with heme-ligand water), -7.3 kcal/mol (without ligand water)/-5.8 kcal/mol (experiment)], whereas the binding of MF was negligible, with only marginal binding binding free energy of -1.7 kcal/mol with 2-MF bound [-3.8 kcal/mol (experiment)], both with and without retained heme-ligand water. Analysis of the free energy components reveal that hydrophobic/nonpolar contributions account for approximately 90% of the total binding free energy of these substrates and are the source of their differential and temperature-dependent CYP2B4 binding. The results indicate the underlying origins of the experimentally observed differential binding affinities of BP and MF, and indicate the plausibility of the use of models derived from moderate sequence identity templates in conjunction with approximate free energy methods in the estimation of ligand-P450 binding affinities.     

Glycogen phosphorylase inhibitors: a free energy perturbation analysis of glucopyranose spirohydantoin analogues

GP catalyzes the phosphorylation of glycogen to Glc-1-P. Because of its fundamental role in the metabolism of glycogen, GP has been the target for a systematic structure-assisted design of inhibitory compounds, which could be of value in the therapeutic treatment of type 2 diabetes mellitus. The most potent catalytic-site inhibitor of GP identified to date is spirohydantoin of glucopyranose (hydan). In this work, we employ MD free energy simulations to calculate the relative binding affinities for GP of hydan and two spirohydantoin analogues, methyl-hydan and n-hydan, in which a hydrogen atom is replaced by a methyl- or amino group, respectively. The results are compared with the experimental relative affinities of these ligands, estimated by kinetic measurements of the ligand inhibition constants. The calculated binding affinity for methyl-hydan (relative to hydan) is 3.75 +/- 1.4 kcal/mol, in excellent agreement with the experimental value (3.6 +/- 0.2 kcal/mol). For n-hydan, the calculated value is 1.0 +/- 1.1 kcal/mol, somewhat smaller than the experimental result (2.3 +/- 0.1 kcal/mol). A free energy decomposition analysis shows that hydan makes optimum interactions with protein residues and specific water molecules in the catalytic site. In the other two ligands, structural perturbations of the active site by the additional methyl- or amino group reduce the corresponding binding affinities. The computed binding free energies are sensitive to the preference of a specific water molecule for two well-defined positions in the catalytic site. The behavior of this water is analyzed in detail, and the free energy profile for the translocation of the water between the two positions is evaluated. The results provide insights into the role of water molecules in modulating ligand binding affinities. A comparison of the interactions between a set of ligands and their surrounding groups in X-ray structures is often used in the interpretation of binding free energy differences and in guiding the design of new ligands. For the systems in this work, such an approach fails to estimate the order of relative binding strengths, in contrast to the rigorous free energy treatment.     

Helix-capping interaction in λ cro protein: A free energy simulation analysis

The stability mutant Tyr-26 → Asp was studied in the Cro protein from bacteriophage λ using free energy molecular dynamics simulations. The mutant was calculated to be more stable than the wild type by 3.0 ± 1.7 kcal/mol/monomer, in reasonable agreement with experiment (1.4 kcal/mol/monomer). Moreover, the aspartic acid in the mutant was found to form a capping interation with the amino terminus of the third α-helix of Cro. The simulations were analyzed to understand better the source of the stability of this helix-capping interaction and to examine the results in light of previous explanations of stabilizing helix caps-namely, a model of local unsatisfied hydrogen bonds at the helix termini and the helix macro dipole model. Analysis of the simulations shows that the stabilizing effect of this charged helical cap is due both to favorable hydrogen bonds with backbone NH groups at the helix terminus and to favorable electrostatic interactions (but not hydrogen bonds) with their carbonyls (effectively the next row of local dipoles in the helix). However, electrostatic interactions are weak or negligible with backbone dipolar groups in the helix further away from the terminus. Moreover, the importance of other local electrostatic interactions with polar side chains near the helix terminus, which are neglected in most treatments of this effect, are shown to be important. Thus, the results support a model that is intermediate between the two previous explanations: both unsatisfied hydrogen bonds at the helix terminus and other, local preoriented dipolar groups stabilize the helix cap. These findings suggest that similar interactions with preoriented dipolar groups may be important for cooperativity in other charge–dipole interactions and may be employed to advantage for molecular design. © 1994 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.     

The severity of osteogenesis imperfecta: a comparison to the relative free energy differences of collagen model peptides

Molecular dynamics simulations were carried out to calculate free energy differences between the folded and unfolded states of wild type and mutant collagen model peptides. The calculated stability of the collagen models was compared with the severity of osteogenesis imperfecta. Free energy differences of Gly → Xaa (Xaa: Ser, Cys, Glu, and Asp) mutations between the wild type and the mutants at position 15 of the model peptide were 3.8, 4.2, 5.6, and 8.8 kcal/mol, respectively. The corresponding free energy differences of a second Gly mutation at the same position in different chains were, on average, 1.3, 1.5, 2.9, and 5.4 kcal/mol, respectively. Free energy simulations were also performed to estimate the relative stability between an oxidized form and a reduced form of the mutants containing two Cys residues, which indicated that the mutant of the collagen-like peptide containing an intramolecular disulfide bond was more stable than the mutant containing one Cys residue but less stable than the wild type. The calculated free energy differences between an oxidized and a reduced form of the mutants containing two Cys residues are 0.8 and 2.6 kcal/mol for the disulfide bonds between Chains A and B and between Chains A and C, respectively.     

The pH-dependent stability of wild-type and mutant transthyretin oligomers

A reduction in pH is known to induce the disassociation of the tetrameric form of transthyretin and favor the formation of amyloid fibers. Using continuum electrostatic techniques, we calculate the titration curves and the stability of dimer and tetramer formation of transthyretin as a function of pH. We find that the tetramer and the dimer become less stable than the monomer as the pH is lowered. The free energy difference is 13.8 kcal/mol for dimer formation and 27 kcal/mol for tetramer formation, from the monomers, when the pH is lowered from 7 to 3.9. Similar behavior is observed for both the wild-type and the mutant protein. Certain residues (namely Glu-72, His-88, His-90, Glu-92, and Tyr-116), play an important role in the binding process, as seen by the considerable pK(1/2) change of these residues upon dimer formation.     

Probing carbohydrate product expulsion from a processive cellulase with multiple absolute binding free energy methods

Understanding the enzymatic mechanism that cellulases employ to degrade cellulose is critical to efforts to efficiently utilize plant biomass as a sustainable energy resource. A key component of cellulase action on cellulose is product inhibition from monosaccharide and disaccharides in the product site of cellulase tunnel. The absolute binding free energy of cellobiose and glucose to the product site of the catalytic tunnel of the Family 7 cellobiohydrolase (Cel7A) of Trichoderma reesei (Hypocrea jecorina) was calculated using two different approaches: steered molecular dynamics (SMD) simulations and alchemical free energy perturbation molecular dynamics (FEP/MD) simulations. For the SMD approach, three methods based on Jarzynski's equality were used to construct the potential of mean force from multiple pulling trajectories. The calculated binding free energies, -14.4 kcal/mol using SMD and -11.2 kcal/mol using FEP/MD, are in good qualitative agreement. Analysis of the SMD pulling trajectories suggests that several protein residues (Arg-251, Asp-259, Asp-262, Trp-376, and Tyr-381) play key roles in cellobiose and glucose binding to the catalytic tunnel. Five mutations (R251A, D259A, D262A, W376A, and Y381A) were made computationally to measure the changes in free energy during the product expulsion process. The absolute binding free energies of cellobiose to the catalytic tunnel of these five mutants are -13.1, -6.0, -11.5, -7.5, and -8.8 kcal/mol, respectively. The results demonstrated that all of the mutants tested can lower the binding free energy of cellobiose, which provides potential applications in engineering the enzyme to accelerate the product expulsion process and improve the efficiency of biomass conversion.     

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.     

Self-association mode of a flavoenzyme D-amino acid oxidase from hog kidney. II. Stoichiometry of holoenzyme association and energetics of subunit association

The self-association pattern of D-amino acid oxidase holoenzyme in 0.1 M sodium pyrophosphate, pH 8.3, at 25 degrees C was examined by the low-angle laser light-scattering method. As to the results of nonlinear least-squares analysis of the apparent weight-average molecular weight (Mwapp) versus protein concentration (c) data, the following three models fitted equally well the data over the concentration range of 0.03-11.4 mg/ml: 1) the model of isodesmic indefinite self-association of the monomer where the dimerization constant differs from the isodesmic association constant, 2) the model which involves the dimerization of the monomer and isodesmic indefinite self-association of the dimer, and 3) the model which involves the trimerization of the monomer and isodesmic indefinite self-association of the trimer. In a more limited concentration range (0.3-11.4 mg/ml), a model of isodesmic indefinite self-association of the stable dimer where the dimer does not dissociate into the monomers cannot be excluded from the above three models. Measurements with the concentration range lowered to 0.03 mg/ml enabled us to exclude unequivocally the model involving such a stable dimer and to extrapolate the Mwapp data to the Mr of the monomer at infinite dilution as in the case of the apoenzyme. The observed sedimentation boundary profiles were qualitatively consistent with the idealized boundary profiles calculated with the model which involves the dimerization of the monomer and isodesmic indefinite self-association of the dimer, so this model is the most probable of the models examined. These results provide the first evidence that the association mode of the holoenzyme is different from that of the apoenzyme, i.e. isodesmic indefinite self-association of the monomer (Tojo, H., Horiike, K., Shiga, K., Nishina, Y., Watari, H., and Yamano, T. (1985) J. Biol. Chem. 260, 12607-12614). The overall linkage scheme, between binding of coenzyme FAD and subunit association, was considered, and the overall free energy change in each process in the scheme was calculated. The total stabilization energies of the intersubunit interaction in the holoenzyme relative to the apoenzyme were found to be -2.2 kcal/mol at the dimerization step and -0.5 kcal/mol at the step of the addition of the dimer to any 2i-mer (i = 1,2, ...).     

Calculations of free energy profiles for the staphylococcal nuclease catalyzed reaction

Calculations of the free energy profile for the first two (rate-limiting) steps of the staphylococcal nuclease catalyzed reaction are reported. The calculations are based on the empirical valence bond method in combination with free energy perturbation molecular dynamics simulations. The calculated activation free energy is in good agreement with experimental kinetic data, and the catalytic effect of the enzyme is reproduced without any arbitrary adjustment of parameters. The enormous reduction of the activation barrier (relative to the reference reaction in water) appears to be largely associated with the strong electrostatic effect of the Ca2+ ion and the two arginine residues in the active site. This favorable electrostatic environment reduces the cost of the general-base catalysis step by almost 15 kcal/mol (by stabilizing the OH- nucleophile) and then stabilizes the developing negative charge on the 5'-phosphate group in the second step of the reaction by about 19 kcal/mol. The basic features of the originally postulated enzyme mechanism (Cotton et al., 1979) are found to be compatible with the observed activation free energy. However, the proposed modification of the mechanism (Sepersu et al., 1987), in which Arg 87 interacts only with the pentacoordinated transition state, is supported by the simulations. Further calculations on the D21E mutant also give results in good agreement with kinetic data.     

Insights into the interactions between HIV-1 integrase and human LEDGF/p75 by molecular dynamics simulation and free energy calculation

Human cellular protein LEDGF/p75 (lens epithelium-derived growth factor) is an important binding partner of human immunodeficiency virus type 1 (HIV-1) integrase (IN). Without LEDGF/p75, HIV-1 can not complete its life cycle. To study the detailed interactions between LEDGF/p75 and HIV-1 IN, and then obtain the hotspots at the binding interface, 13 ns molecular dynamics simulations were carried out here. One-hundred snapshots extracted from the last 4 ns trajectories were used for calculation of binding free energy and decomposition of the energy by residue. First, the structural changes and their dynamic interactions were investigated focused on the production stage. And then, the free energy was discussed. On the basis of the above results, it could be suggested that residues Gln168, Glu170, and Thr174 in chain A of IN, Thr125, and Trp131 in chain B of IN as well as Ile365, Asp366, Phe406, and Val408 in LEDGF/p75 were responsible for their binding. These results might be helpful for discovery and design of small molecules to interrupt the interaction between HIV-1 IN and LEDGF/p75.     

On the calculation of binding free energies using continuum methods: Application to MHC class I protein-peptide interactions

This paper describes a methodology to calculate the binding free energy (ΔG) of a protein-ligand complex using a continuum model of the solvent. A formal thermodynamic cycle is used to decompose the binding free energy into electrostatic and non-electrostatic contributions. In this cycle, the reactants are discharged in water, associated as purely nonpolar entities, and the final complex is then recharged. The total electrostatic free energies of the protein, the ligand, and the complex in water are calculated with the finite difference Poisson-Boltzmann (FDPB) method. The nonpolar (hydrophobic) binding free energy is calculated using a free energy-surface area relationship, with a single alkane/water surface tension coefficient (γ_aw). The loss in backbone and side-chain configurational entropy upon binding is estimated and added to the electrostatic and the nonpolar components of ΔG. The methodology is applied to the binding of the murine MHC class I protein H-2K^b with three distinct peptides, and to the human MHC class I protein HLA-A2 in complex with five different peptides. Despite significant differences in the amino acid sequences of the different peptides, the experimental binding free energy differences (ΔΔG_exp) are quite small (<0.3 and <2.7 kcal/mol for the H-2K^b and HLA-A2 complexes, respectively). For each protein, the calculations are successful in reproducing a fairly small range of values for ΔΔG_calc (<4.4 and <5.2 kcal/mol, respectively) although the relative peptide binding affinities of H-2K^b and HLA-A2 are not reproduced. For all protein-peptide complexes that were treated, it was found that electrostatic interactions oppose binding whereas nonpolar interactions drive complex formation. The two types of interactions appear to be correlated in that larger nonpolar contributions to binding are generally opposed by increased electrostatic contributions favoring dissociation. The factors that drive the binding of peptides to MHC proteins are discussed in light of our results.     

Insights into the functional role of protonation states in the HIV-1 protease-BEA369 complex: molecular dynamics simulations and free energy calculations

The molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method combined with molecular dynamics (MD) simulations were used to investigate the functional role of protonation in human immunodeficiency virus type 1 (HIV-1) protease complexed with the inhibitor BEA369. Our results demonstrate that protonation of two aspartic acids (Asp25/Asp25′) has a strong influence on the dynamics behavior of the complex, the binding free energy of BEA369, and inhibitor–residue interactions. Relative binding free energies calculated using the MM-PBSA method show that protonation of Asp25 results in the strongest binding of BEA369 to HIV-1 protease. Inhibitor–residue interactions computed by the theory of free energy decomposition also indicate that protonation of Asp25 has the most favorable effect on binding of BEA369. In addition, hydrogen-bond analysis based on the trajectories of the MD simulations shows that protonation of Asp25 strongly influences the water-mediated link of a conserved water molecule, Wat301. We expect that the results of this study will contribute significantly to binding calculations for BEA369, and to the design of high affinity inhibitors.     

Accurate prediction of the binding free energy and analysis of the mechanism of the interaction of replication protein A (RPA) with ssDNA

The eukaryotic replication protein A (RPA) has several pivotal functions in the cell metabolism, such as chromosomal replication, prevention of hairpin formation, DNA repair and recombination, and signaling after DNA damage. Moreover, RPA seems to have a crucial role in organizing the sequential assembly of DNA processing proteins along single stranded DNA (ssDNA). The strong RPA affinity for ssDNA, K(A) between 10(-9)-10(-10) M, is characterized by a low cooperativity with minor variation for changes on the nucleotide sequence. Recently, new data on RPA interactions was reported, including the binding free energy of the complex RPA70AB with dC(8) and dC(5), which has been estimated to be -10 ± 0.4 kcal mol(-1) and -7 ± 1 kcal mol(-1), respectively. In view of these results we performed a study based on molecular dynamics aimed to reproduce the absolute binding free energy of RPA70AB with the dC(5) and dC(8) oligonucleotides. We used several tools to analyze the binding free energy, rigidity, and time evolution of the complex. The results obtained by MM-PBSA method, with the use of ligand free geometry as a reference for the receptor in the separate trajectory approach, are in excellent agreement with the experimental data, with ±4 kcal mol(-1) error. This result shows that the MM-PB(GB)SA methods can provide accurate quantitative estimates of the binding free energy for interacting complexes when appropriate geometries are used for the receptor, ligand and complex. The decomposition of the MM-GBSA energy for each residue in the receptor allowed us to correlate the change of the affinity of the mutated protein with the ΔG(gas+sol) contribution of the residue considered in the mutation. The agreement with experiment is optimal and a strong change in the binding free energy can be considered as the dominant factor in the loss for the binding affinity resulting from mutation.     

Interaction of lipid-free apolipoprotein A-I with cholesterol revealed by molecular modeling

We report the modeling of the interaction of differently self-associated lipid-free apoA-I with cholesterol monomer and tail-to-tail (TT) or face-to-face (FF) cholesterol dimer. Cholesterol dimerization is exploited to reconcile the existing experimental data on cholesterol binding to apoA-I with extremely low critical micelle concentration of cholesterol. Two crystal structures of 1–43 N-truncated apolipoprotein Δ(1-43)A-I tetramer (PDB ID: 1AV1, structure B), 185–243 C-truncated apolipoprotein Δ(185-243)A-I dimer (PDB ID: 3R2P, structure M) were analyzed. Cholesterol monomers bind to multiple binding sites in apoA-I monomer, dimer and tetramer with low, moderate and high energy (?10 to ?28 kJ/mol with Schrödinger package), still insufficient to overcome the thermodynamic restriction by cholesterol micellization (?52.8 kJ/mol). The binding sites partially coincide with the putative cholesterol-binding motifs. However, apoA-I monomer and dimer existing in structure B, that contain nonoverlapping and non-interacting pairs of binding sites with high affinity for TT and FF cholesterol dimers, can bind in common 14 cholesterol molecules that correspond to existing values. ApoA-I monomer and dimer in structure M can bind in common 6 cholesterol molecules. The values of respective total energy of cholesterol binding up to 64.5 and 67.0 kJ/mol for both B and M structures exceed the free energy of cholesterol micellization. We hypothesize that cholesterol dimers may simultaneously interact with extracellular monomer and dimer of lipid-free apoA-I, that accumulate at acid pH in atheroma. The thermodynamically allowed apolipoprotein-cholesterol interaction outside the macrophage may represent a new mechanism of cholesterol transport by apoA-I from atheroma, in addition to ABCA1-mediated cholesterol efflux.     

A binding free energy hot spot in the ankyrin repeat protein GABPbeta mediated protein-protein interaction

The frequently observed ankyrin repeat motif represents a structural scaffold evolved for mediating protein-protein interactions. As such, these repeats modulate a diverse range of cellular functions. We thermodynamically characterized the heterodimeric GA-binding protein (GABP) alphabeta complex and focused specifically on the interaction mediated by the ankyrin repeat domain of the GABPbeta. Our isothermal titration calorimetric analysis of the interaction between the GABP subunits determined an association constant (K(A)) of 6.0 x 10(8) M(-1) and that the association is favorably driven by a significant change in enthalpy (DeltaH) and a minor change in entropy (-TDeltaS). A total of 16 GABPbeta interface residues were chosen for alanine scanning mutagenesis. The calorimetrically measured differences in the free energy of binding were compared to computationally calculated values resulting in a correlation coefficient r = 0.71. We identified three spatially contiguous hydrophobic and aromatic residues that form a binding free energy hot spot (DeltaDeltaG > 2.0 kcal/mol). One residue provides structural support to the hot spot residues. Three non-hot spot residues are intermediate contributors (DeltaDeltaG approximately 1.0 kcal/mol) and create a canopy-like structure over the hot spot residues to possibly occlude solvent and orientate the subunits. The remaining interface residues are located peripherally and have weak contributions. Finally, our mutational analysis revealed a significant entropy-enthalpy compensation for this interaction.     

Sequence context modulates the stability of a GxxxG-mediated transmembrane helix-helix dimer

To quantify the relationship between sequence and transmembrane dimer stability, a systematic mutagenesis and thermodynamic study of the protein-protein interaction residues in the glycophorin A transmembrane helix-helix dimer was carried out. The results demonstrate that the glycophorin A transmembrane sequence dimerizes when its GxxxG motif is abolished by mutation to large aliphatic residues, suggesting that the sequence encodes an intrinsic propensity to self-associate independent of a GxxxG motif. In the presence of an intact GxxxG motif, the glycophorin A dimer stability can be modulated over a span of -0.5 kcal mol(-1) to +3.2 kcal mol(-1) by mutating the surrounding sequence context. Thus, these flanking residues play an active role in determining the transmembrane dimer stability. To assess the structural consequences of the thermodynamic effects of mutations, molecular models of mutant transmembrane domains were constructed, and a structure-based parameterization of the free energy change due to mutation was carried out. The changes in association free energy for glycophorin A mutants can be explained primarily by changes in packing interactions at the protein-protein interface. The energy cost of removing favorable van der Waals interactions was found to be 0.039 kcal mol(-1) per A2 of favorable occluded surface area. The value corresponds well with estimates for mutations in bacteriorhodopsin as well as for those mutations in the interiors of soluble proteins that create packing defects.