Energetics of ligand-induced conformational flexibility in the lactose permease of Escherichia coli

Isothermal titration calorimetry has been applied to characterize the thermodynamics of ligand binding to wild-type lactose permease (LacY) and a mutant (C154G) that strongly favors an inward facing conformation. The affinity of wild-type or mutant LacY for ligand and the change in free energy (DeltaG) upon binding are similar. However, with the wild type, the change in free energy upon binding is due primarily to an increase in the entropic free energy component (TDeltaS), whereas in marked contrast, an increase in enthalpy (DeltaH) is responsible for DeltaG in the mutant. Thus, wild-type LacY behaves as if there are multiple ligand-bound conformational states, whereas the mutant is severely restricted. The findings also indicate that the structure of the mutant represents a conformational intermediate in the overall transport cycle.     

The role of backbone motions in ligand binding to the c-Src SH3 domain.

The Src homology 3 (SH3) domain of pp60(c-src) (Src) plays dual roles in signal transduction, through stabilizing the repressed form of the Src kinase and through mediating the formation of activated signaling complexes. Transition of the Src SH3 domain between a variety of binding partners during progression through the cell cycle requires adjustment of a delicate free energy balance. Although numerous structural and functional studies of SH3 have provided an in-depth understanding of structural determinants for binding, the origins of binding energy in SH3-ligand interactions are not fully understood. Considering only the protein-ligand interface, the observed favorable change in standard enthalpy (DeltaH=-9.1 kcal/mol) and unfavorable change in standard entropy (TDeltaS=-2.7 kcal/mol) upon binding the proline-rich ligand RLP2 (RALPPLPRY) are inconsistent with the predominantly hydrophobic interaction surface. To investigate possible origins of ligand binding energy, backbone dynamics of free and RLP2-bound SH3 were performed via (15)N NMR relaxation and hydrogen-deuterium (H/(2)H) exchange measurements. On the ps-ns time scale, assuming uncorrelated motions, ligand binding results in a significant reduction in backbone entropy (-1.5(+/-0.6) kcal/mol). Binding also suppresses motions on the micros-ms time scale, which may additionally contribute to an unfavorable change in entropy. A large increase in protection from H/(2)H exchange is observed upon ligand binding, providing evidence for entropy loss due to motions on longer time scales, and supporting the notion that stabilization of pre-existing conformations within a native state ensemble is a fundamental paradigm for ligand binding. Observed changes in motion on all three time scales occur at locations both near and remote from the protein-ligand interface. The propagation of ligand binding interactions across the SH3 domain has potential consequences in target selection through altering both free energy and geometry in intact Src, and suggests that looking beyond the protein-ligand interface is essential in understanding ligand binding energetics.     

Thermodynamic characterization of affinity maturation: the D1.3 antibody and a higher-affinity mutant

Understanding the structural and dynamic determinants of binding free energy in the antigen-antibody bond is of great interest. Much work has focused on selective mutations in order to locate key interaction residues, but this generally results in reduced affinity. The present work instead examines a higher-affinity mutant to characterize the thermodynamic pathway of the affinity maturation process. We have compared the antigen binding energetics of scFv D1.3, an anti-hen egg lysozyme single chain antibody, with a higher-affinity mutant (Hawkins, R. E., Russell, S. J., Baier, M. and Winter, G. (1993). J. Mol. Biol. 234, 958-964). The mutant has five-fold higher affinity for lysozyme but nearly the same enthalpy and heat capacity change upon binding, as measured by isothermal titration calorimetry. Thus, much of the binding free energy difference can be attributed to entropic effects. Fluorescence quenching with acrylamide indicates that this more favorable entropy change may result from a more flexible mutant-lysozyme complex and thus be a configurational entropy effect.     

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.     

Biophysical insights into the interaction of clofazimine with human alpha 1-acid glycoprotein: a multitechnique approach

Alpha1-acid glycoprotein (AAG) is a major acute phase protein of human plasma. Binding of clofazimine to AAG is investigated using optical spectroscopy and molecular docking tools. We found significant quenching of intrinsic fluorescence of AAG upon the binding of clofazimine, binding mode is static with binding constant of 3.52 × 10⁴at 298 K. The Gibbs free energy change is found to be negative for the interaction of clofazimine with AAG indicating spontaneity of the binding process. Binding of clofazimine induced ordered structure in protein and lead to molecular compaction. Molecular docking results indicate the binding site is located in the central beta barrel, hydrogen bonding and hydrophobic interactions are main bonding forces between AAG–clofazimine.     

Understanding the molecular basis of MK2-p38α signaling complex assembly: insights into protein-protein interaction by molecular dynamics and free energy studies

The formation of a p38 MAPK and MAPK-activated protein kinase 2 (MK2) signaling complex is physiologically relevant to cellular responses such as the proinflammatory cytokine production. The interaction between p38α isoform and MK2 is of great importance for this signaling. In this study, molecular dynamics simulation and binding free energy calculation were performed on the MK2-p38α signaling complex to investigate the protein-protein interaction between the two proteins. Dynamic domain motion analyses were performed to analyze the conformational changes between the unbound and bound states of proteins during the interaction. The activation loop, αF-I helices, and loops among α helices in the C-lobe of MK2 are found to be highly flexible and exhibit significant changes upon p38α binding. The results also show that after the binding of p38α, the N- and C-terminal domains of MK2 display an opening and twisting motion centered on the activation loop. The molecular mechanics Poisson-Boltzmann and generalized-Born surface area (MM-PB/GBSA) methods were used to calculate binding free energies between MK2 and p38α. The analysis of the components of binding free energy calculation indicates that the van der Waals interaction and the nonpolar solvation energy provide the driving force for the binding process, while the electrostatic interaction contributes critically to the specificity, rather than to MK2-p38α binding affinity. The contribution of each residue at the interaction interface to the binding affinity of MK2 with p38α was also analyzed by free energy decomposition. Several important residues responsible for the protein-protein interaction were also identified.     

Thermodynamics of the alpha-helix-coil transition of amphipathic peptides in a membrane environment: implications for the peptide-membrane binding equilibrium

Amphipathic alpha-helices are the membrane binding motif in many proteins. The corresponding peptides are often random coil in solution but are folded into an alpha-helix upon interaction with the membrane. The energetics of this ubiquitous folding process are still a matter of conjecture. Here, we present a new method to quantitatively analyze the thermodynamics of peptide folding at the membrane interface. We have systematically varied the helix content of a given amphipathic peptide when bound to the membrane and have correlated the thermodynamic binding parameters determined by isothermal titration calorimetry with the alpha-helix content obtained by circular dichroism spectroscopy. The peptides investigated were the antibiotic magainin 2 amide and three analogs in which two adjacent amino acid residues were substituted by their d-enantiomers. The thermodynamic parameters controlling the alpha-helix formation were found to be linearly related to the helicity of the membrane-bound peptides. Helix formation at the membrane surface is characterized by an enthalpy change of DeltaH(helix) approximately -0.7 kcal/mol per residue, an entropy change of DeltaS(helix) approximately -1.9 cal/molK residue and a free energy change of DeltaG(helix)=-0.14 kcal/mol residue. Helix formation is a strong driving force of peptide insertion into the membrane and accounts for about 50 % of the free energy of binding. An increase in temperature entails an unfolding of the membrane-bound helix. The temperature dependence can be described with the Zimm-Bragg theory and the enthalpy of unfolding agrees with that deduced from isothermal titration calorimetry.     

Antiviral drug design: computational analyses of the effects of the L100I mutation for HIV-RT on the binding of NNRTIs

Monte Carlo/free energy perturbation (MC/FEP) calculations were used to evaluate the binding free energy change for HIV-RT/inhibitor complexes upon L100I mutation. Inhibitor size and flexibility adjacent to hydrogen-bonding sites are evident as important considerations for antiviral drug design.     

Structure and thermodynamics of RNA-protein binding: using molecular dynamics and free energy analyses to calculate the free energies of binding and conformational change

An adaptive binding mechanism, requiring large conformational rearrangements, occurs commonly with many RNA-protein associations. To explore this process of reorganization, we have investigated the conformational change upon spliceosomal U1A-RNA binding with molecular dynamics (MD) simulations and free energy analyses. We computed the energetic cost of conformational change in U1A-hairpin and U1A-internal loop binding using a hybrid of molecular mechanics and continuum solvent methods. Encouragingly, in all four free energy comparisons (two slightly different proteins, two different RNAs), the free macromolecule was more stable than the bound form by the physically reasonable value of approximately 10 kcal/mol. We calculated the absolute binding free energies for both complexes to be in the same range as that found experimentally.     

Ranking ligand binding affinities with avidin: a molecular dynamics-based interaction energy study.

The binding of 14 biotin analogues to avidin is examined to evaluate the viability of calculating binding free energy based on molecular dynamics (MD) trajectories. Two approaches were investigated in this work. The first one uses the linear interaction energy approximation, while the other approach utilizes the interaction free energy. The results obtained from these two methods were found to correlate well with the experimental binding free energy data for 10 out of 14 ligands. For the other four ligands, both methods overestimate their binding strength by more than 7 kcal/mol. Free energy calculations using the thermodynamic integration method are employed to understand this overestimation. The effect of protein flexibility on binding free energy calculation and the effect of charged or neutral ligands on the calculated results are discussed. MD simulations are shown to be able to provide insight into the interactions occurring in the active site and the origins of variations in binding free energy.     

Binding of ibuprofen to human hemoglobin: elucidation of their molecular recognition by spectroscopy,calorimetry, and molecular modeling techniques

Ibuprofen, used for the treatment of acute and chronic pain, osteoarthritis, rheumatoid arthritis, and related conditions has ample affinity to globular proteins. Here we have explored this fundamental study pertaining to the interaction of ibuprofen with human hemoglobin (HHb), using multispectroscopic, calorimetric, and molecular modeling techniques to gain insights into molecular aspects of binding mechanism. Ibuprofen-induced graded decrease in absorption spectra indicates protein disruption along with sedimentation of HHb particle. Red shifting of absorption peak at 195 nm indicates alteration in the secondary structure of HHb upon interaction with ibuprofen. Flouremetric and isothermal titration calorimetric (ITC) studies suggested one binding site in HHb for ibuprofen at 298.15 K. However, with increase in temperature, ITC revealed increasing number of binding sites. The negative values of Gibbs energy change (ΔG⁰) and enthalpy change (ΔH⁰) along with positive value of entropy change (ΔS⁰) strongly suggest that it is entropy-driven spontaneous exothermic reaction. Moreover, hydrophobic interaction, hydrogen bonding, and π–π interaction play major role in this binding process as evidenced from ANS (8-anilino-1-napthalenesulphonic acid), sucrose binding, and molecular modeling studies. The interaction impacts on structural integrity and functional aspects of HHb as confirmed by CD spectroscopy, increased free iron release, increased rate of co-oxidation and decreased rate of esterase activity. These findings suggest us to conclude that ibuprofen upon interaction perturbs both structural and functional aspects of HHb.     

Free energy decomposition of protein-protein interactions.

A free energy decomposition scheme has been developed and tested on antibody-antigen and protease-inhibitor binding for which accurate experimental structures were available for both free and bound proteins. Using the x-ray coordinates of the free and bound proteins, the absolute binding free energy was computed assuming additivity of three well-defined, physical processes: desolvation of the x-ray structures, isomerization of the x-ray conformation to a nearby local minimum in the gas-phase, and subsequent noncovalent complex formation in the gas phase. This free energy scheme, together with the Generalized Born model for computing the electrostatic solvation free energy, yielded binding free energies in remarkable agreement with experimental data. Two assumptions commonly used in theoretical treatments; viz., the rigid-binding approximation (which assumes no conformational change upon complexation) and the neglect of vdW interactions, were found to yield large errors in the binding free energy. Protein-protein vdW and electrostatic interactions between complementary surfaces over a relatively large area (1400--1700 A(2)) were found to drive antibody-antigen and protease-inhibitor binding.     

A calorimetric investigation of the interaction of the lac repressor with inducer

A calorimetric study has been made of the interaction between the lac repressor and isopropyl-1-thio-beta-D-galactopyranoside (IPTG). The buffer-corrected enthalpy of reaction at 25 degrees C was found to be -15.6, -24.7, -4.6 kJ/mol of bound IPTG at pH 7.0, pH 8.1, and pH 9.0, respectively. This large range of enthalpy values is in contrast to a maximum difference in the free energy of the reaction of only 1.5 kJ/mol of bound IPTG between these pH values. The reaction was found by calorimetric measurements in different buffers to be accompanied by an uptake of 0.29 mol of protons/mol of bound IPTG at pH 8.1. The pH dependency of the reaction enthalpy suggests differences in the extent of protonation of the binding site and the involvement of H bonding with IPTG. The lack of strong hydrophobic contributions in the IPTG binding process is revealed by the absence of any determinable heat capacity change for the reaction at pH 7.0. The presence of phosphate buffer significantly alters the enthalpy of IPTG binding at higher pH values, but has little effect upon the binding constant. This implies that highly negative phosphate species change the nature of the IPTG binding site without any displacement of phosphate upon IPTG binding.     

Two-step mechanism of binding of apolipoprotein E to heparin: implications for the kinetics of apolipoprotein E-heparan sulfate proteoglycan complex formation on cell surfaces

The interaction of apolipoprotein E (apoE) with cell-surface heparan sulfate proteoglycans is an important step in the uptake of lipoprotein remnants by the liver. ApoE interacts predominantly with heparin through the N-terminal binding site spanning the residues around 136-150. In this work, surface plasmon resonance analysis was employed to investigate how amphipathic alpha-helix properties and basic residue organization in this region modulate binding of apoE to heparin. The apoE/heparin interaction involves a two-step process; apoE initially binds to heparin with fast association and dissociation rates, followed by a step exhibiting much slower kinetics. Circular dichroism and surface plasmon resonance experiments using a disulfide-linked mutant, in which opening of the N-terminal helix bundle was prevented, demonstrated that there is no major secondary or tertiary structural change in apoE upon heparin binding. Mutations of Lys-146, a key residue for the heparin interaction, greatly reduced the favorable free energy of binding of the first step without affecting the second step, suggesting that electrostatic interaction is involved in the first binding step. Although lipid-free apoE2 tended to bind less than apoE3 and apoE4, there were no significant differences in rate and equilibrium constants of binding among the apoE isoforms in the lipidated state. Discoidal apoE3-phospholipid complexes using a substitution mutant (K143R/K146R) showed similar binding affinity to wild type apoE3, indicating that basic residue specificity is not required for the effective binding of apoE to heparin, unlike its binding to the low density lipoprotein receptor. In addition, disruption of the alpha-helix structure in the apoE heparin binding region led to an increased favorable free energy of binding in the second step, suggesting that hydrophobic interactions contribute to the second binding step. Based on these results, it seems that cell-surface heparan sulfate proteoglycan localizes apoE-enriched remnant lipoproteins to the vicinity of receptors by fast association and dissociation.     

Quantum and molecular dynamics study for binding of macrocyclic inhibitors to human alpha-thrombin

Molecular dynamics simulations followed by quantum mechanical calculation and Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) analysis have been carried out to study binding of proline- and pyrazinone-based macrocyclic inhibitors (L86 and T76) to human alpha-thrombin. Detailed binding interaction energies between these inhibitors and individual protein fragments are calculated using DFT method based on a new quantum mechanical approach for computing protein-ligand interaction energy. The analysis of detailed interaction energies provides insight on the protein-ligand binding mechanism. Study shows that T76 and L86 bind to thrombin in a very similar "inhibition mode" except that T76 has relatively weaker binding interaction with Glu(217). The analysis from quantum calculation of binding interaction is consistent with the MM-PBSA calculation of binding free energy, and the calculated free energies for L86/T76-thrombin binding agree well with the experimental data.     

Solvation change and ion release during aminoacylation by aminoacyl-tRNA synthetases

Discrimination between cognate and non-cognate tRNAs by aminoacyl-tRNA synthetases occurs at several steps of the aminoacylation pathway. We have measured changes of solvation and counter-ion distribution at various steps of the aminoacylation pathway of glutamyl- and glutaminyl-tRNA synthetases. The decrease in the association constant with increasing KCl concentration is relatively small for cognate tRNA binding when compared to known DNA–protein interactions. The electro-neutral nature of the tRNA binding domain may be largely responsible for this low ion release stoichiometry, suggesting that a relatively large electrostatic component of the DNA–protein interaction free energy may have evolved for other purposes, such as, target search. Little change in solvation upon tRNA binding is seen. Non-cognate tRNA binding actually increases with increasing KCl concentration indicating that charge repulsion may be a significant component of binding free energy. Thus, electrostatic interactions may have been used to discriminate between cognate and non-cognate tRNAs in the binding step. The catalytic constant of glutaminyl-tRNA synthetase increases with increasing osmotic pressure indicating a water release of 8.4 ± 1.4 mol/mol in the transition state, whereas little change is seen in the case of glutamyl-tRNA synthetase. We propose that the significant amount of water release in the transition state, in the case of glutaminyl-tRNA synthetase, is due to additional contact of the protein with the tRNA in the transition state.     

Electrostatic contributions to the binding free energy of the lambdacI repressor to DNA.

A model based on the nonlinear Poisson-Boltzmann (NLPB) equation is used to study the electrostatic contribution to the binding free energy of the lambdacI repressor to its operator DNA. In particular, we use the Poisson-Boltzmann model to calculate the pKa shift of individual ionizable amino acids upon binding. We find that three residues on each monomer, Glu34, Glu83, and the amino terminus, have significant changes in their pKa and titrate between pH 4 and 9. This information is then used to calculate the pH dependence of the binding free energy. We find that the calculated pH dependence of binding accurately reproduces the available experimental data over a range of physiological pH values. The NLPB equation is then used to develop an overall picture of the electrostatics of the lambdacI repressor-operator interaction. We find that long-range Coulombic forces associated with the highly charged nucleic acid provide a strong driving force for the interaction of the protein with the DNA. These favorable electrostatic interactions are opposed, however, by unfavorable changes in the solvation of both the protein and the DNA upon binding. Specifically, the formation of a protein-DNA complex removes both charged and polar groups at the binding interface from solvent while it displaces salt from around the nucleic acid. As a result, the electrostatic contribution to the lambdacI repressor-operator interaction opposes binding by approximately 73 kcal/mol at physiological salt concentrations and neutral pH. A variety of entropic terms also oppose binding. The major force driving the binding process appears to be release of interfacial water from the protein and DNA surfaces upon complexation and, possibly, enhanced packing interactions between the protein and DNA in the interface. When the various nonelectrostatic terms are described with simple models that have been applied previously to other binding processes, a general picture of protein/DNA association emerges in which binding is driven by the nonpolar interactions, whereas specificity results from electrostatic interactions that weaken binding but are necessary components of any protein/DNA complex.     

Thermodynamics of the folding of D-glyceraldehyde-3-phosphate dehydrogenase assisted by protein disulfide isomerase studied by microcalorimetry.

Thermodynamics of the refolding of denatured D-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) assisted by protein disulfide isomerase (PDI), a molecular chaperone, has been studied by isothermal microcalorimetry at different molar ratios of PDI/GAPDH and temperatures using two thermodynamic models proposed for chaperone-substrate binding and chaperone-assisted substrate folding, respectively. The binding of GAPDH folding intermediates to PDI is driven by a large favorable enthalpy decrease with a large unfavorable entropy reduction, and shows strong enthalpy-entropy compensation and weak temperature dependence of Gibbs free energy change. A large negative heat-capacity change of the binding, -156 kJ.mol(-1).K(-1), at all temperatures examined indicates that hydrophobic interaction is a major force for the binding. The binding stoichiometry shows one dimeric GAPDH intermediate per PDI monomer. The refolding of GAPDH assisted by PDI is a largely exothermic reaction at 15.0-25.0 degrees C. With increasing temperature from 15.0 to 37.0 degrees C, the PDI-assisted reactivation yield of denatured GAPDH upon dilution decreases. At 37.0 degrees C, the spontaneous reactivation, PDI-assisted reactivation and intrinsic molar enthalpy change during the PDI-assisted refolding of GAPDH are not detected.