Thermodynamics Of Ligand Binding Research Articles

The Dynamic Nature of RNA as Key to Understanding Riboswitch Mechanisms

Riboswitches are gene regulation elements within RNA that recognize specific metabolites. They predominantly occur in the untranslated leader regions of bacterial messenger RNA (mRNA). Upon metabolite binding to the aptamer domain, a structural change in the adjoining downstream expression platform signals "on" or "off" for gene expression. Researchers have achieved much progress in characterizing ligand-bound riboswitch states at the molecular level; an impressive number of high-resolution structures of aptamer-ligand complexes is now available. These structures have significantly contributed toward our understanding of how riboswitches interact with their natural ligands and with structurally related analogues. In contrast, relatively little is known about the nature of the unbound (apo) form of riboswitches. Moreover, the details of how changes in the aptamer domain are transduced into conformational changes in the decision-making expression platform remain murky. In this Account, we report on recent efforts aimed at the characterization of free states, ligand recognition, and ligand-induced folding in riboswitches. Riboswitch action is best approached as a cotranscriptional process, which implies sequential folding and release of the aptamer prior to the signaling of the expression platform. Thus, a complex interplay of several factors has to be taken into account, such as speed of transcription, transcriptional pausing, kinetics and thermodynamics of RNA structure formation, and kinetics and thermodynamics of ligand binding. The response mechanism appears to be best described as a process in which ligand recognition critically dictates the folding pathway of the nascent mRNA during its expression; the resulting structures determine the interactions with the transcriptional or translational apparatus. We discuss experimental methods that offer insight into the dynamics of the free riboswitch state. These include probing experiments, such as in-line and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) techniques, small-angle X-ray scattering (SAXS) analysis, NMR spectroscopy, and fluorescence spectroscopy, including single-molecule fluorescence resonance energy transfer (smFRET) imaging. One of our research contributions is an approach, termed 2ApFold, that incorporates noninvasive 2-aminopurine modifications in riboswitches. The fluorescence response of these moieties is used to delineate the order of secondary-tertiary structure formation and rearrangements taking place during ligand-induced folding. This information can be used to explore the kinetics of ligand recognition and to analyze the degree of structure preorganization of the free riboswitch state. Furthermore, we discuss a recent smFRET study on the SAM-II riboswitch; this report underscores the importance of choosing strategic labeling patterns that leave maximal conformational freedom to the regulatory interaction. Finally, we comment on how riboswitch ligand recognition appeals to the concepts of conformational selection and induced fit, and on the question of whether riboswitches act under thermodynamic or kinetic control. This Account highlights the fact that a thorough understanding of RNA dynamics in vitro is required to shed light on cellular riboswitch mechanisms. Elucidating these mechanisms will contribute not only to ongoing efforts to target riboswitches with antibiotics but also to attempts to engineer artificial cell regulation systems.

Mechanism and thermodynamics of ligand binding to auxin amidohydrolase

BrILL2 is catalytically the most efficient auxin amidohydrolase from Brassica rapa, playing a key role in auxin metabolism by catalyzing its release from amino acid conjugates. Auxins, with the most abundant representative indole-acetic acid ([1H-indol-3-yl]-acetic acid, IAA), are a group of plant hormones that in very small concentrations regulate ubiquitin-mediated degradation of transcription regulators. Kinetic studies on BrILL2 showed that it hydrolyzes alanine conjugates of IAA and of its larger analogues, indole-propionic acid (3-[1H-indol-3-yl]-propionic acid, IPA) and indole-butyric acid (4-[1H-indol-3-yl]-butyric acid, IBA). Structurally, BrILL2 belongs to the largest known family of metallopeptidases (M20) that share a recognizable 3D structure, characterized by two perpendicular domains. Its members have been implicated in numerous biochemical processes and have been found across all species sequenced to date. Here, molecular dynamics simulations were carried out to study structural and thermodynamic properties of ligand binding to BrILL2. A conformational change was captured in multiple copies of 10 ns long simulations, described by a rigid body movement of the two domains, and its associated key interactions between residues were examined. For the three substrates, complexes in two possible binding modes were recreated, along with a single binding mode for the putative substrate tryptophanyl-alanine (Trp-Ala), which were subsequently simulated in multiple copies of 10 ns long simulations. Thermodynamic calculations were used to assess their binding affinities and explain the selectivity toward the longer ligands. Based on the results, a possible route for the reaction is proposed.

Structure and Function of Cytochromes P450 2B: From Mechanism-Based Inactivators to X-Ray Crystal Structures and Back

This article reviews work from the author dating back to 1978 and focuses on the structural basis of cytochrome P450 (P450) function using available contemporary techniques. Early studies used mechanism-based inactivators that bound to the protein moiety of hepatic P450s to try to localize the active site. Subsequent studies used cDNA cloning, heterologous expression, site-directed mutagenesis, and homology modeling based on multiple bacterial P450 X-ray crystal structures to predict the active sites of CYP2B enzymes with considerable accuracy. Breakthroughs in engineering and expression of mammalian P450s enabled us to determine our first X-ray crystal structure of ligand-free rabbit CYP2B4. To date, we have solved 11 CYP2B4 and three human CYP2B6 structures, which represent four significantly different conformations. The plasticity of CYP2B4 has been confirmed by deuterium exchange mass spectrometry and is substantiated by molecular dynamics simulations. In addition to major movement of secondary structure elements, more subtle reorientation of active site side chains, especially Phe206, Phe297, and Glu301, contributes to the ability of CYP2B enzymes to bind various ligands. Isothermal titration calorimetry has proven to be a useful tool for studying the thermodynamics of ligand binding to CYP2B4 and CYP2B6, and NMR has enabled study of ligand binding orientation in solution as an adjunct to X-ray crystallography. A major challenge remains to harness the power of the various approaches to facilitate prediction of CYP2B specificity and inhibition.

Thermodynamics and Solvent Effects on Substrate and Cofactor Binding in Escherichia coli Chromosomal Dihydrofolate Reductase

Chromosomal dihydrofolate reductase from Escherichia coli catalyzes the reduction of dihydrofolate to tetrahydrofolate using NADPH as a cofactor. The thermodynamics of ligand binding were examined using an isothermal titration calorimetry approach. Using buffers with different heats of ionization, zero to a small, fractional proton release was observed for dihydrofolate binding, while a proton was released upon NADP(+) binding. The role of water in binding was additionally monitored using a number of different osmolytes. Binding of NADP(+) is accompanied by the net release of ∼5-24 water molecules, with a dependence on the identity of the osmolyte. In contrast, binding of dihydrofolate is weakened in the presence of osmolytes, consistent with "water uptake". Different effects are observed depending on the identity of the osmolyte. The net uptake of water upon dihydrofolate binding was previously observed in the nonhomologous R67-encoded dihydrofolate reductase (dfrB or type II enzyme) [Chopra, S., et al. (2008) J. Biol. Chem. 283, 4690-4698]. As R67 dihydrofolate reductase possesses a nonhomologous sequence and forms a tetrameric structure with a single active site pore, the observation of weaker DHF binding in the presence of osmolytes in both enzymes implicates cosolvent effects on free dihydrofolate. Consistent with this analysis, stopped flow experiments find betaine mostly affects DHF binding via changes in k(on), while betaine mostly affects NADPH binding via changes in k(off). Finally, nonadditive enthalpy terms when binary and ternary cofactor binding events are compared suggest the presence of long-lived conformational transitions that are not included in a simple thermodynamic cycle.

Thermodynamics of ligand binding and efficiency.

Analysis of the experimental binding thermodynamics for approximately 100 protein-ligand complexes provides important insights into the factors governing ligand affinity and efficiency. The commonly accepted correlation between enthalpy and -TΔS is clearly observed for this relatively diverse data set. It is also clear that affinity (i.e., ΔG) is not generally correlated to either enthalpy or -TΔS. This is a worrisome trend since the vast majority of computational structure-based design is carried out using interaction energies for one, or at most a few, ligand poses. As such, these energies are most closely comparable to enthalpies not free energies. Closer inspection of the data shows that in a few cases the enthalpy (or -TΔS) is correlated with free energy. It is tempting to speculate that this could be an important consideration as to why some targets are readily amenable to modeling and others are not. Additionally, analysis of the enthalpy and -TΔS efficiencies shows that the trends observed for ligand efficiencies with respect to molecular size are primarily a consequence of enthalpic, not entropic, effects.

Determination of the volume changes induced by ligand binding to heat shock protein 90 using high-pressure denaturation

The volume changes accompanying ligand binding to proteins are thermodynamically important and could be used in the design of compounds with specific binding properties. Measuring the volumetric properties could yield as much information as the enthalpic properties of binding. Pressure-based methods are significantly more laborious than temperature methods and are underused. Here we present a pressure shift assay (PressureFluor, analogous to the ThermoFluor thermal shift assay) that uses high pressure to denature proteins. The PressureFluor method was used to study the ligand binding thermodynamics of heat shock protein 90 (Hsp90). Ligands stabilize the protein against pressure denaturation, similar to the stabilization against temperature denaturation. The equations that relate the ligand dosing, protein concentration, and binding constant with the volumes and compressibilities of unfolding and binding are presented.

Determination of the Volume Changes Induced by Ligand Binding to Hsp90 Using High Pressure Denaturation

The volume changes accompanying ligand binding to proteins are thermodynamically important and could be used in the design of compounds with specific binding properties. Measuring the volumetric properties could yield as much information, as the enthalpic properties of binding. Pressure-based methods are significantly more laborious than temperature methods and are underused. Here, we present a pressure shift assay (PressureFluor, analogous to the ThermoFluor, thermal shift assay) that uses high pressure to denature proteins. The PressureFluor method was used to study the ligand binding thermodynamics of heat shock protein 90 (Hsp90).Increased concentration of a ligand caused the shift in the Pm as shown in the Figure. The volumes of the protein in the absence and presence of several ligands were determined. The compressibilities of the system could be also determined with significantly lower precision than the volumes. The formulas describing the relationship between the added ligand concentration and the Pm were derived and will be presented.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Protein flexibility and conformational entropy in ligand design targeting the carbohydrate recognition domain of galectin-3.

Rational drug design is predicated on knowledge of the three-dimensional structure of the protein−ligand complex and the thermodynamics of ligand binding. Despite the fundamental importance of both enthalpy and entropy in driving ligand binding, the role of conformational entropy is rarely addressed in drug design. In this work, we have probed the conformational entropy and its relative contribution to the free energy of ligand binding to the carbohydrate recognition domain of galectin-3. Using a combination of NMR spectroscopy, isothermal titration calorimetry, and X-ray crystallography, we characterized the binding of three ligands with dissociation constants ranging over 2 orders of magnitude. 15N and 2H spin relaxation measurements showed that the protein backbone and side chains respond to ligand binding by increased conformational fluctuations, on average, that differ among the three ligand-bound states. Variability in the response to ligand binding is prominent in the hydrophobic core, where a distal cluster of methyl groups becomes more rigid, whereas methyl groups closer to the binding site become more flexible. The results reveal an intricate interplay between structure and conformational fluctuations in the different complexes that fine-tunes the affinity. The estimated change in conformational entropy is comparable in magnitude to the binding enthalpy, demonstrating that it contributes favorably and significantly to ligand binding. We speculate that the relatively weak inherent protein−carbohydrate interactions and limited hydrophobic effect associated with oligosaccharide binding might have exerted evolutionary pressure on carbohydrate-binding proteins to increase the affinity by means of conformational entropy.

On the interaction of luminol with human serum albumin: Nature and thermodynamics of ligand binding

The mechanism and thermodynamic parameters for the binding of luminol (LH 2) with human serum albumin was explored by steady state and picosecond time-resolved fluorescence spectroscopy. It was shown that out of two possible LH 2 conformers present is solution, only one is accessible for binding with HSA. The thermodynamic parameters like enthalpy (Δ H) and entropy (Δ S) change corresponding to the ligand binding process were also estimated by performing the experiment at different temperatures. The ligand replacement experiment with bilirubin confirms that LH 2 binds into the sub-domain IIA of the protein.

Long-Timescale Molecular-Dynamics Simulations of the Major Urinary Protein Provide Atomistic Interpretations of the Unusual Thermodynamics of Ligand Binding

The mouse major urinary protein (MUP) has proved to be an intriguing test bed for detailed studies on protein-ligand recognition. NMR, calorimetric, and modeling investigations have revealed that the thermodynamics of ligand binding involve a complex interplay between competing enthalpic and entropic terms. We performed six independent, 1.2 μs molecular-dynamics simulations on MUP—three replicates on the apo-protein, and three on the complex with the pheromone isobutylmethoxypyrazine. Our findings provide the most comprehensive picture to date of the structure and dynamics of MUP, and how they are modulated by ligand binding. The mechanical pathways by which amino acid side chains can transmit information regarding ligand binding to surface loops and either increase or decrease their flexibility (entropy-entropy compensation) are identified. Dewetting of the highly hydrophobic binding cavity is confirmed, and the results reveal an aspect of ligand binding that was not observed in earlier, shorter simulations: bound ligand retains extensive rotational freedom. Both of these features have significant implications for interpretations of the entropic component of binding. More generally, these simulations test the ability of current molecular simulation methods to produce a reliable and reproducible picture of protein dynamics on the microsecond timescale.

Comparison of Entropic Contributions to Binding in a “Hydrophilic” versus “Hydrophobic” Ligand−Protein Interaction

In the present study we characterize the thermodynamics of binding of histamine to recombinant histamine-binding protein (rRaHBP2), a member of the lipocalin family isolated from the brown-ear tick Rhipicephalus appendiculatus. The binding pocket of this protein contains a number of charged residues, consistent with histamine binding, and is thus a typical example of a “hydrophilic” binder. In contrast, a second member of the lipocalin family, the recombinant major urinary protein (rMUP), binds small hydrophobic ligands, with a similar overall entropy of binding in comparison with rRaHBP2. Having extensively studied ligand binding thermodynamics for rMUP previously, the data we obtained in the present study for HBP enables a comparison of the driving forces for binding between these classically distinct binding processes in terms of entropic contributions from ligand, protein, and solvent. In the case of rRaHBP2, we find favorable entropic contributions to binding from desolvation of the ligand; however, the overall entropy of binding is unfavorable due to a dominant unfavorable contribution arising from the loss of ligand degrees of freedom, together with the sequestration of solvent water molecules into the binding pocket in the complex. This contrasts with binding in rMUP where desolvation of the protein binding pocket makes a minor contribution to the overall entropy of binding given that the pocket is substantially desolvated prior to binding.

Implications of fast-time scale dynamics of human DNA/RNA cytosine methyltransferases (DNMTs) for protein function

The role of protein dynamics in the control of substrate recognition, catalysis, and protein–protein interactions is often underestimated. Recently, a number of studies have examined the contribution of protein dynamics to the thermodynamics of ligand binding in detail, mostly using NMR relaxation measurements and molecular dynamics (MD) simulations. The results unequivocally demonstrate that conformational dynamics play a pivotal role in the properties and functions of proteins, and ignoring this contribution is likely to lead to substantial errors when explaining the biological function of proteins and in predictions of the binding affinities of their cognate ligands. However, the details of the interplay between structure and dynamics and the way it affects the biological function of the target protein remain poorly understood. In this study, the changes in fast (picosecond-to-nanosecond time scale) dynamics of catalytic domains of four human cytosine DNA methyltransferases (DNMTs) were studied using molecular dynamics (MD) simulations. The results provide insight into the protein dynamics changes that occur upon binding of the cofactor, S-adenosylmethionine (SAM). Contrary to expectations, increased amplitude of motions of backbone amide (N–H) and terminal heavy atom (C–C) bond vectors was observed in all studied DNMTs upon binding of SAM. These results imply that the cofactor binding causes a global increase in the extent of protein dynamics in the short time scale. This global dynamic change constitutes a favourable entropic contribution to the free energy of SAM binding. These results suggest that cytosine DNA methyltransferases may exploit changes in their fast scale dynamics to reduce the entropic cost of the substrate binding.

Special Series: Advances in the investigation of the structure‐function relationship in nucleic acids and their assemblies

Special Series: Advances in the investigation of the structure‐function relationship in nucleic acids and their assemblies

Kinetics and Thermodynamics of Ligand Binding to a Molten Globular Enzyme and Its Native Counterpart

An engineered monomeric chorismate mutase (mMjCM) has been found to combine high catalytic activity with the characteristics of a molten globule. To gain insight into the dramatic structural changes that accompany binding of a transition-state analog, we examined mMjCM by isothermal calorimetry and compared it with its dimeric parent protein, MjCM (CM from Methanococcus jannaschii), a thermostable and conventionally folded enzyme. As expected for a ligand-induced ordering process, there is a large entropic penalty for binding to the monomer relative to the dimer (−TΔΔS=5.1±0.5 kcal/mol, at 20 °C). However, this unfavorable entropy term is largely offset by enthalpic gains (ΔΔH=−3.5±0.4 kcal/mol), presumably arising from tightening of non-covalent interactions throughout the monomeric complex. Stopped-flow kinetic measurements further reveal that the catalytic molten globule binds and releases ligands significantly faster than its natural counterpart, demonstrating that partial structural disorder can speed up molecular recognition. These results illustrate how structural plasticity may strongly perturb the thermodynamics and kinetics of transition-state recognition while negligibly affecting catalytic efficiency.

Role of the active-site solvent in the thermodynamics of factor Xa ligand binding.

Understanding the underlying physics of the binding of small-molecule ligands to protein active sites is a key objective of computational chemistry and biology. It is widely believed that displacement of water molecules from the active site by the ligand is a principal (if not the dominant) source of binding free energy. Although continuum theories of hydration are routinely used to describe the contributions of the solvent to the binding affinity of the complex, it is still an unsettled question as to whether or not these continuum solvation theories describe the underlying molecular physics with sufficient accuracy to reliably rank the binding affinities of a set of ligands for a given protein. Here we develop a novel, computationally efficient descriptor of the contribution of the solvent to the binding free energy of a small molecule and its associated receptor that captures the effects of the ligand displacing the solvent from the protein active site with atomic detail. This descriptor quantitatively predicts (R(2) = 0.81) the binding free energy differences between congeneric ligand pairs for the test system factor Xa, elucidates physical properties of the active-site solvent that appear to be missing in most continuum theories of hydration, and identifies several features of the hydration of the factor Xa active site relevant to the structure-activity relationship of its inhibitors.

Dynamic Allostery in the Ring Protein TRAP

We have discovered distinct, characteristic differences in the thermodynamic signatures of tryptophan binding by trp RNA-binding attenuation protein (TRAP) from two different bacterial species. The TRAP 11mer ring binds 11 molecules of tryptophan at symmetry-related sites. Tryptophan binding to Bacillus stearothermophilus TRAP is not cooperative, but isothermal titration calorimetry shows that filling the first tryptophan binding sites of Bacillus subtilis TRAP has a marked effect on the thermodynamics of subsequent ligand binding. We have identified a single, conservative amino acid replacement (Ile to Leu) in B. subtilis TRAP that abolishes this effect, and suggest the initial ligand binding causes a change throughout the wild-type protein ring.

Thermodynamics of Ligand Binding to P450 2B4 and P450eryF Studied by Isothermal Titration Calorimetry

Structural plasticity and cooperativity in ligand recognition are two key aspects of the catalytic diversity of cytochrome P450 enzymes. As more mammalian P450 crystal structures have emerged, computational modeling has become a major tool to predict drug metabolism and interactions. There is a need for real solution thermodynamic data to support modeling and crystallographic observations. Using isothermal titration calorimetry (ITC) we successfully evaluated the conformational flexibility of P450 2B4 in binding imidazole inhibitors of different size and chemistry and dissected the stoichiometry and energetics of ligand binding allostery in P450eryF. Thermodynamic signatures obtained by ITC nicely correlated with structural and modeling results. Thus, ITC is a powerful tool to study structure-function relationships in P450s.

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.

Kinetics and Thermodynamics of Ligand Binding by Cytochrome P450 3A4

Cytochrome P450 (P450) 3A4, the major catalyst involved in human drug oxidation, displays substrate- and reaction-dependent homotropic and heterotropic cooperative behavior. Although several models have been proposed, these mainly rely on steady-state kinetics and do not provide information on the contribution of the individual steps of P450 catalytic cycle to the observed cooperativity. In this work, we focused on the kinetics of substrate binding, and the fluorescent properties of bromocriptine and alpha-naphthoflavone allowed analysis of an initial ligand-P450 3A4 interaction that does not cause a perturbation of the heme spectrum. The binding stoichiometry for bromocriptine was determined to be unity using isothermal titration calorimetry and equilibrium dialysis methods, suggesting that the ligand bound to the peripheral site during the initial encounter dissociates subsequently. A three-step substrate binding model is proposed, based on absorbance and fluorescence stopped-flow kinetic data and equilibrium binding data obtained with bromocriptine, and evaluated using kinetic modeling. The results are consistent with the substrate molecule binding at a site peripheral to the active site and subsequently moving toward the active site to bind to the heme and resulting in a low to high spin iron shift. The last step is attributed to a conformational change in the enzyme active site. The later steps of binding were shown to have rate constants comparable with the subsequent steps of the catalytic cycle. The P450 3A4 binding process is more complex than a two-state system, and the overlap of rates of some of the events with subsequent steps is proposed to underlie the observed cooperativity.

Kinetics and thermodynamics of ligand binding by cytochrome P450 3A4 and relevance to substrate cooperativity

Cytochrome P450 (P450) 3A4, the major catalyst involved in human drug oxidation, displays substrate and reaction dependent cooperative behavior. Although several models have been proposed, these mainly rely on steady-state kinetics and do not provide information on the contribution of the individual steps of P450 catalytic cycle to the observed cooperativity. In this work, we focused on the kinetics of substrate binding, and the fluorescent properties of bromocriptine and α-naphthoflavone allowed analysis of an initial ligand-P450 3A4 interaction that does not cause a perturbation of the heme spectrum. A three-step substrate binding model is proposed, based on absorbance and fluorescence stopped-flow kinetic data, and evaluated using kinetic modeling. The results are consistent with the first substrate molecule binding at a site peripheral to the active site, followed by a second ligand molecule binding to the heme. The last step is attributed to a conformational change in the enzyme active site. The binding stoichiometry for bromocriptine was determined to be unity using isothermal titration calorimetry and equilibrium dialysis methods, suggesting that the ligand bound to the peripheral site during the initial encounter may subsequently dissociate. The later steps of binding were shown to have rate constants comparable to the subsequent steps of the catalytic cycle. The P450 3A4 binding process is more complex than a two-state system and the overlap of rates of some of the events with subsequent steps is proposed to underlie the observed cooperativity. Supported in part by USPHS R01 CA90426 and P30 ES00267