Thermodynamics of T-cell receptor-peptide/MHC interactions: progress and opportunities

alphabeta T-cell receptors (TCRs) recognize peptide antigens presented by class I or class II major histocompatibility complex molecules (pMHC). Here we review the use of thermodynamic measurements in the study of TCR-pMHC interactions, with attention to the diversity in binding thermodynamics and how this is related to the variation in TCR-pMHC interfaces. We show that there is no enthalpic or entropic signature for TCR binding; rather, enthalpy and entropy changes vary in a compensatory manner that reflects a narrow free energy window for the interactions that have been characterized. Binding enthalpy and entropy changes do not correlate with structural features such as buried surface area or the number of hydrogen bonds within TCR-pMHC interfaces, possibly reflecting the myriad of contributors to binding thermodynamics, but likely also reflecting a reliance on van't Hoff over calorimetric measurements and the unaccounted influence of equilibria linked to binding. TCR-pMHC binding heat capacity changes likewise vary considerably. In some cases, the heat capacity changes are consistent with conformational differences between bound and free receptors, but there is little data indicating these conformational differences represent the need to organize disordered CDR loops. In this regard, we discuss how thermodynamics may provide additional insight into conformational changes occurring upon TCR binding. Finally, we highlight opportunities for the further use of thermodynamic measurements in the study of TCR-pMHC interactions, not only for understanding TCR binding in general, but also for understanding specifics of individual interactions and the engineering of TCRs with desired molecular recognition properties.     

Thermodynamic benchmark study using Biacore technology

A total of 22 individuals participated in this benchmark study to characterize the thermodynamics of small-molecule inhibitor-enzyme interactions using Biacore instruments. Participants were provided with reagents (the enzyme carbonic anhydrase II, which was immobilized onto the sensor surface, and four sulfonamide-based inhibitors) and were instructed to collect response data from 6 to 36 degrees C. van't Hoff enthalpies and entropies were calculated from the temperature dependence of the binding constants. The equilibrium dissociation and thermodynamic constants determined from the Biacore analysis matched the values determined using isothermal titration calorimetry. These results demonstrate that immobilization of the enzyme onto the sensor surface did not alter the thermodynamics of these interactions. This benchmark study also provides insights into the opportunities and challenges in carrying out thermodynamic studies using optical biosensors.     

The interactions of nucleic acids at elevated hydrostatic pressure

The application of elevated hydrostatic pressure on the order of a few thousand bar provides insight into the molecular forces responsible for stabilizing the conformations and non-covalent interactions of biological molecules in aqueous solution. In particular, the parameters derived from these studies have enabled researchers to glean information regarding the importance of hydration in the energetics and kinetics of these systems. This review presents data concerned with the application of hydrostatic pressure to study the thermodynamics, kinetics, and structure of nucleic acids and the interactions between nucleic acids and proteins, enzymes, and drugs. These complexes often form extremely stable non-covalent complexes in which electrostatic interactions play an important role. The sensitivity of these interactions to pressure offers a valuable experimental tool for investigating the energetics of the complexes.     

SIMPLE estimate of the free energy change due to aliphatic mutations: superior predictions based on first principles

The bioinformatics revolution of the last decade has been instrumental in the development of empirical potentials to quantitatively estimate protein interactions for modeling and design. Although computationally efficient, these potentials hide most of the relevant thermodynamics in 5-to-40 parameters that are fitted against a large experimental database. Here, we revisit this longstanding problem and show that a careful consideration of the change in hydrophobicity, electrostatics, and configurational entropy between the folded and unfolded state of aliphatic point mutations predicts 20-30% less false positives and yields more accurate predictions than any published empirical energy function. This significant improvement is achieved with essentially no free parameters, validating past theoretical and experimental efforts to understand the thermodynamics of protein folding. Our first principle analysis strongly suggests that both the solute-solute van der Waals interactions in the folded state and the electrostatics free energy change of exposed aliphatic mutations are almost completely compensated by similar interactions operating in the unfolded ensemble. Not surprisingly, the problem of properly accounting for the solvent contribution to the free energy of polar and charged group mutations, as well as of mutations that disrupt the protein backbone remains open.     

Structure and energetics of protein-protein interactions: the role of conformational heterogeneity in OMTKY3 binding to serine proteases

Proteins with flexible binding surfaces can interact with numerous binding partners. However, this promiscuity is more difficult to understand in "rigid-body" proteins, whose binding results in little, or no, change in the position of backbone atoms. The binding of Kazal inhibitors to serine proteases is considered a classic case of rigid-body binding, although they bind to a wide range of proteases. We have studied the thermodynamics of binding of the Kazal serine protease inhibitor, turkey ovomucoid third domain (OMTKY3), to the serine protease subtilisin Carlsberg using isothermal titration calorimetry and have determined the crystal structure of the complex at very high resolution (1.1A). Comparison of the binding energetics and structure to other OMTKY3 interactions demonstrates that small changes in the position of side-chains can make significant contributions to the binding thermodynamics, including the enthalpy of binding. These effects emphasize that small, "rigid-body" proteins are still dynamic structures, and these dynamics make contributions to both the enthalpy and entropy of binding interactions.     

Cooperativity in macromolecular assembly

The thermodynamic principle of cooperativity is used to drive the formation of specific macromolecular complexes during the assembly of a macromolecular machine. Understanding cooperativity provides insight into the mechanisms that govern assembly and disassembly of multicomponent complexes. Our understanding of assembly mechanisms is lagging considerably behind our understanding of the structure and function of these complexes. A significant challenge remains in tackling the thermodynamics and kinetics of the intermolecular interactions required for all cellular functions.     

Isothermal titration calorimetry of metal ions binding to proteins: An overview of recent studies

Isothermal titration calorimetry (ITC) is a technique that is capable of quantifying the stoichiometry, equilibrium constants and thermodynamics of molecular binding events. Thus, important information about the interaction of metal ions with biological macromolecules can be obtained with ITC measurements. This review highlights many of the recent studies of metal ions binding to proteins that have used ITC to quantify the thermodynamics of metal-protein interactions.     

Thermodynamics of sequence-specific protein-DNA interactions

The molecular recognition processes in sequence-specific protein-DNA interactions are complex. The only feature common to all sequence-specific protein-DNA structures is a large interaction interface, which displays a high degree of complementarity in terms of shape, polarity and electrostatics. Many molecular mechanisms act in concert to form the specific interface. These include conformational changes in DNA and protein, dehydration of surfaces, reorganization of ion atmospheres, and changes in dynamics. Here we review the current understanding of how different mechanisms contribute to the thermodynamics of the binding equilibrium and the stabilizing effect of the different types of noncovalent interactions found in protein-DNA complexes. The relation to the thermodynamics of small molecule-DNA binding and protein folding is also briefly discussed.     

Force spectroscopy of single DNA and RNA molecules

Experiments in which single molecules of RNA and DNA are stretched, and the resulting force as a function of extension is measured have yielded new information about the physical, chemical and biological properties of these important molecules. The behavior of both single-stranded and double-stranded nucleic acids under changing solution conditions, such as ionic strength, pH and temperature, has been studied in detail. There has also been progress in using these techniques to study both the kinetics and equilibrium thermodynamics of DNA-protein interactions. These studies generate unique insights into the functions of these proteins in the cell.     

Relaxation and electrophoretic effects in polyelectrolyte solutions. I. Salt-free solutions

Relations between relaxation and electrophoretic effects in salt-free polyelectrolyte solutions are studied in terms of nonequilibrium thermodynamics and of binary friction coefficients. The significance and the method of determining these coefficients are explained. Consideration of experimental results and of long- and short-range interactions suggests that the concept of “ion condensation” is needed to provide a consistent desciption of transport processes for linear flexible polyelectrolytes. Empirical relations previously used are shown to have only limited validity.     

Kinetic analysis of the binding of Ulex europeas agglutinin 1 (UEA 1) to H-antigenic fucolipid

The kinetics and thermodynamics of Ulex europeas agglutinin 1-H-antigenic determinant was studied by monitoring the binding of the lectin to H-antigenic fucolipid incorporated in liposomes tethered to the sensor surface by surface plasmon resonance. The second-order rate constants for the interactions are several order of magnitude slower than the diffusion controlled reactions. Analyses of activation parameters reveals that these slow second-order rate constants are a consequence of an unfavorable activation enthalpy term.     

Thermodynamics of metal ion binding. 2. Metal ion binding by carbonic anhydrase variants

The ability to construct molecular motifs with predictable properties in aqueous solution requires an extensive knowledge of the relationships between structure and energetics. The design of metal binding motifs is currently an area of intense interest in the bioorganic community. To date synthetic motifs designed to bind metal ions lack the remarkable affinities observed in biological systems. To better understand the structural basis of metal ion affinity, we report here the thermodynamics of binding of divalent zinc ions to wild-type and mutant carbonic anhydrases and the interpretation of these parameters in terms of structure. Mutations were made both to the direct His ligand at position 94 and to indirect, or second-shell, ligands Gln-92, Glu-117, and Thr-199. The thermodynamics of ligand binding by several mutant proteins is complicated by the development of a second zinc binding site on mutation; such effects must be considered carefully in the interpretation of thermodynamic data. In all instances modification of the protein produces a complex series of changes in both the enthalpy and entropy of ligand binding. In most cases these effects are most readily rationalized in terms of ligand and protein desolvation, rather than in terms of changes in the direct interactions of ligand and protein. Alteration of second-shell ligands, thought to function primarily by orienting the direct ligands, produces profoundly different effects on the enthalpy of binding, depending on the nature of the residue. These results suggest a range of activities for these ligands, contributing both enthalpic and entropic effects to the overall thermodynamics of binding. Together, our results demonstrate the importance of understanding relationships between structure and hydration in the construction of novel ligands and biological polymers.     

Thermodynamics of aliphatic and aromatic hydrocarbons in water

Makhatadze and Privalov have analyzed the thermodynamics of transfer of aliphatic and aromatic hydrocarbons from the gas phase into water. Finding that the hydration free energy of aliphatic and aromatic hydrocarbons have different signs, they conclude that the mechanism causing hydrophobicity of these solutes is of a different nature. Here, we offer an alternative analysis of the dissolution of these non-polar compounds into water based on a recently published interpretation scheme for thermodynamic transfer functions. Our analysis shows that the hydrophobicity of aromatic and aliphatic hydrocarbons is qualitatively the same, i.e. its causes are the same namely the extremely high cohesive energy of water which overcomes the favorable solute-solute and solute-water interactions. However, both analyses conclude that the experimentally observed quantitative difference between the interactions of water with aliphatic and aromatic hydrocarbons, can be assigned to the formation of aromatic ring-water H-bonds.     

Anion binding to a protein-protein complex lacks dependence on net charge

The binding of anions to proteins occurs in numerous physiological and metabolic processes. In an effort to understand the factors important in these interactions, we have studied the weak binding of phosphate and sulfate to a protein-protein complex using isothermal titration calorimetry. To our knowledge, this is the first system in which the thermodynamics of anion binding have been determined calorimetrically. By studying both phosphate and sulfate binding and using a range of pH values, the charge on the anion was varied from approximately -1 to -2. Surprisingly, no dependence of the binding energetics on the charge of the anion was observed. This result indicates that charge-charge interactions are not the dominant factor in binding and suggests the importance of hydrogen bonding in specifically recognizing and coordinating anions.     

The ABRF-MIRG’02 Study: Assembly State,Thermodynamic, and Kinetic Analysis of an Enzyme/Inhibitor Interaction

Fully characterizing the interactions involving biomolecules requires information on the assembly state, affinity, kinetics, and thermodynamics associated with complex formation. The analytical technologies often used to measure biomolecular interactions include analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). In order to evaluate the capabilities of core facilities to implement these technologies, the Association of Biomolecular Resource Facilities (ABRF) Molecular Interactions Research Group (MIRG) developed a standardized model system and distributed it to a panel of AUC, ITC, and SPR operators. The model system was composed of a well-characterized enzyme-inhibitor pair, namely bovine carbonic anhydrase II (CA II) and 4-carboxybenzenesulfonamide (CBS). Study participants were asked to measure one or more of the following: (1) the molecular mass, homogeneity, and assembly state of CA II by AUC; (2) the affinity and thermodynamics for complex formation by ITC; and (3) the affinity and kinetics of complex formation by SPR. The results from this study provide a benchmark for comparing the capabilities of individual laboratories and for defining the utility of the different instrumentation.     

NMR methods for studying quadruplex nucleic acids

Solution NMR spectroscopy has traditionally played a central role in examining quadruplex structure, dynamics, and interactions. Here, an overview is given of the methods currently applied to structural, dynamics, thermodynamics, and kinetics studies of nucleic acid quadruplexes and associated cations.     

Thermodynamic Analysis of the Binding of 2F5 (Fab and Immunoglobulin G Forms) to Its gp41 Epitope Reveals a Strong Influence of the Immunoglobulin Fc Region on Affinity

Immunotherapies and vaccines based on the induction of broadly neutralizing monoclonal antibodies (bNAbs) have become outstanding strategies against HIV-1. Diverse bNAbs recognizing different regions of the HIV-1 envelope have been identified and extensively studied. However, there is little information about the thermodynamics of binding of these bNAbs and their epitopes. We used isothermal titration calorimetry to characterize thermodynamically the interactions between bNAb 2F5 (in both the IgG and Fab forms) and its functional and core epitope peptides. We found that these interactions are enthalpically driven and opposed by a negative entropy change. The highest affinity was found for 2F5 IgG for its functional epitope, indicating that additional interactions involving residues flanking the core epitope contribute strongly to higher affinity. In addition, the strong influence of the Fc region on the binding affinity suggests long-range allosteric effects within IgG. Our results provide useful information for developing new therapeutics against HIV-1 and, in a broader scope, contribute to a better understanding of antigen-antibody recognition.     

Enthalpic denaturation of collagen satisfies the general relation of the change in Delta H(d) from Delta C(p) for proteins at zero hydroxyproline level]

One of the noticeable peculiarities of thermodynamics of collagen is an anomalous high magnitude of the enthalpy of denaturation delta Hd at a very low thermostability. Taking into account recent ideas about the role of hydrophobic interactions in determination of the thermodynamic function of protein denaturation, it is shown that the high magnitude of delta Hd of collagen in comparison with those of globular proteins can be explained by two factors: a significant contribution of residues of 4-hydroxyproline and small magnitude of hydrophobic interactions.     

Making cool drugs hot: isothermal titration calorimetry as a tool to study binding energetics

Characterization of the thermodynamics of binding interactions is important in improving our understanding of bimolecular recognition and forms an essential part of the rational drug design process. Isothermal titration calorimetry (ITC) is rapidly becoming established as the method of choice for undertaking such studies. The power of ITC lies in its unique ability to measure binding reactions by the detection of the heat change during the binding interaction. Since heat changes occur during many physicochemical processes, ITC has a broad application, ranging from chemical and biochemical binding studies to more complex processes involving enthalpy changes, such as enzyme kinetics. Several features of ITC have facilitated its preferential use compared to other techniques that estimate affinity. It is a sensitive, rapid, and direct method with no requirement for chemical modification or immobilization. It is the only technique that directly measures enthalpy of binding and so eliminates the need for van't Hoff analysis, which can be time consuming and prone to uncertainty in parameter values. Although ITC has facilitated the measurement of the thermodynamics governing binding reactions, interpretation of these parameters in structural terms is still a major challenge.