Irene Luque

Structural stability of binding sites: consequences for binding affinity and allosteric effects.

During the course of biological function, proteins interact with other proteins, ligands, substrates, inhibitors, etc. These interactions occur at precisely defined locations within the protein but their effects are sometimes propagated to distal regions, triggering highly specific responses. These effects can be used as signals directed to activate or inhibit other sites, modulate interactions with other molecules, and/or establish inter‐molecular communication networks. During the past decade, it has become evident that the energy of stabilization of the protein structure is not evenly distributed throughout the molecule and that, under native conditions, proteins lack global cooperativity and are characterized by the occurrence of multiple independent local unfolding events. From a biological point of view, it is important to assess if this uneven distribution reflects specific functional requirements. For example, are binding sites more likely to be found in well structured regions, unstable regions, or mixed regions? In this article, we have addressed these questions by performing a structure‐based thermodynamic stability analysis of non‐structurally homologous proteins for which high resolution structures of their complexes with specific ligands are available. The results of these studies indicate that for all 16 proteins considered, the binding sites have a dual character and are characterized by the presence of regions with very low structural stability and regions with high stability. In many cases the low stability regions are loops that become stable and cover a significant portion of low molecular weight ligands upon binding. For enzymes, catalytic residues are usually, but not always, located in regions with high structural stability. It is shown that this arrangement provides significant advantages for the optimization of binding affinity of small ligands. In allosteric enzymes, low stability regions in the regulatory site are shown to play a crucial role in the transmission of information to the catalytic site. Proteins 2000;41:63–71.

Structural parameterization of the binding enthalpy of small ligands

A major goal in ligand and drug design is the optimization of the binding affinity of selected lead molecules. However, the binding affinity is defined by the free energy of binding, which, in turn, is determined by the enthalpy and entropy changes. Because the binding enthalpy is the term that predominantly reflects the strength of the interactions of the ligand with its target relative to those with the solvent, it is desirable to develop ways of predicting enthalpy changes from structural considerations. The application of structure/enthalpy correlations derived from protein stability data has yielded inconsistent results when applied to small ligands of pharmaceutical interest (MW < 800). Here we present a first attempt at an empirical parameterization of the binding enthalpy for small ligands in terms of structural information. We find that at least three terms need to be considered: (1) the intrinsic enthalpy change that reflects the nature of the interactions between ligand, target, and solvent; (2) the enthalpy associated with any possible conformational change in the protein or ligand upon binding; and, (3) the enthalpy associated with protonation/deprotonation events, if present. As in the case of protein stability, the intrinsic binding enthalpy scales with changes in solvent accessible surface areas. However, an accurate estimation of the intrinsic binding enthalpy requires explicit consideration of long‐lived water molecules at the binding interface. The best statistical structure/enthalpy correlation is obtained when buried water molecules within 5–7 Å of the ligand are included in the calculations. For all seven protein systems considered (HIV‐1 protease, dihydrodipicolinate reductase, Rnase T1, streptavidin, pp60c‐Src SH2 domain, Hsp90 molecular chaperone, and bovine β‐trypsin) the binding enthalpy of 25 small molecular weight peptide and nonpeptide ligands can be accounted for with a standard error of ±0.3 kcal · mol−1. Proteins 2002;49:181–190.

Structure-based thermodynamic design of peptide ligands: Application to peptide inhibitors of the aspartic protease endothiapepsin

The prediction of binding affinities from structure is a necessary requirement in the development of structure‐based molecular design strategies. In this paper, a structural parameterization of the energetics previously developed in this laboratory has been incorporated into a molecular design algorithm aimed at identifying peptide conformations that minimize the Gibbs energy. This approach has been employed in the design of mutants of the aspartic protease inhibitor pepstatin A. The simplest design strategy involves mutation and/or chain length modification of the wild‐type peptide inhibitor. The structural parameterization allows evaluation of the contribution of different amino acids to the Gibbs energy in the wild‐type structure, and therefore the identification of potential targets for mutation in the original peptide. The structure of the wild‐type complex is used as a template to generate families of conformational structures in which specific residues have been mutated. The most probable conformations of the mutated peptides are identified by systematically rotating around the side‐chain and backbone torsional angles and calculating the Gibbs potential function of each conformation according to the structural parametrization. The accuracy of this approach has been tested by chemically synthesizing two different mutants of pepstatin A. In one mutant, the alanine at position five has been replaced by a phenylalanine, and in the second one a glutamate has been added at the carboxy terminus of pepstatin A. The thermodynamics of association of pepstatin A and the two mutants have been measured experimentally and the results compared with the predictions. The difference between experimental and predicted Gibbs energies for pepstatin A and the two mutants is 0.23 ± 0.06 kcal/mol. The excellent agreement between experimental and predicted values demonstrates that this approach can be used in the optimization of peptide ligands. Proteins 30:74–85, 1998.

The application of thermodynamic methods in drug design

Abstract The optimization of lead compounds as viable drug candidates involves the optimization of their binding affinity towards the selected target. The binding affinity, K a , is determined by the Gibbs energy of binding, Δ G , which in turn is determined by the enthalpy, Δ H , and entropy, Δ S , changes (Δ G =Δ H − T Δ S ). In principle, many combinations of Δ H and Δ S values can give rise to the same Δ G value and, therefore, elicit the same binding affinity. However, enthalpically dominated ligands do not behave the same as entropically dominated ligands. Current paradigms in drug design usually generate highly hydrophobic and conformationally constrained ligands. The thermodynamic signature of these ligands is an entropically dominated binding affinity often accompanied by an unfavorable binding enthalpy. Conformationally constrained ligands cannot easily adapt to changes in the geometry of the binding site, being therefore highly susceptible to drug resistance mutations or naturally occurring genetic polymorphisms. The design of ligands with the capability to adapt to a changing target requires the introduction of certain elements of flexibility or the relaxation of some conformational constraints. Since these compounds pay a larger conformational entropy penalty upon binding, the optimization of their binding affinity requires the presence of a favorable binding enthalpy. In this paper, experimental and computational strategies aimed at identifying and optimizing enthalpic ligands will be discussed and applied to the case of HIV-1 protease inhibitors. It is shown that a thermodynamic guide to drug design permits the identification of drug candidates with a lower susceptibility to target mutations causing drug resistance.

Role of Interfacial Water Molecules in Proline-rich Ligand Recognition by the Src Homology 3 Domain of Abl

The interaction of Abl-Src homology 3 domain (SH3) with the high affinity peptide p41 is the most notable example of the inconsistency existing between the currently accepted description of SH3 complexes and their binding thermodynamic signature. We had previously hypothesized that the presence of interfacial water molecules is partially responsible for this thermodynamic behavior. We present here a thermodynamic, structural, and molecular dynamics simulation study of the interaction of p41 with Abl-SH3 and a set of mutants designed to alter the water-mediated interaction network. Our results provide a detailed description of the dynamic properties of the interfacial water molecules and a molecular interpretation of the thermodynamic effects elicited by the mutations in terms of the modulation of the water-mediated hydrogen bond network. In the light of these results, a new dual binding mechanism is proposed that provides a better description of proline-rich ligand recognition by Abl-SH3 and that has important implications for rational design.

Thermodynamic characterization of the folding equilibrium of the human Nedd4-WW4 domain: at the frontiers of cooperative folding.

WW domains are the smallest naturally independent beta-sheet protein structures available to date and constitute attractive model systems for investigating the determinants of beta-sheet folding and stability. Nonetheless, their small size and low cooperativity pose a difficult challenge for a quantitative analysis of the folding equilibrium. We describe here a comprehensive thermodynamic characterization of the conformational equilibrium of the fourth WW domain from the human ubiquitin ligase Nedd4 (hNedd4-WW4) using a combination of calorimetric and spectroscopic techniques with several denaturing agents (temperature, pH, and chemical denaturants). Our results reveal that even though the experimental data can be described in terms of a two-state equilibrium, spectral data together with anomalous values for some thermodynamic parameters (a strikingly low temperature of maximum stability, a higher than expected native-state heat capacity, and a small specific enthalpy of unfolding) could be indicative of more complex types of equilibria, such as one-state downhill folding or alternative native conformations. Moreover, double-perturbation experiments reveal some features that, in spite of the apparent linear correlation between the thermodynamic parameters, seem to be indicative of a complex conformational equilibrium in the presence of urea. In summary, the data presented here point toward the existence of a low-energy barrier between the different macrostates of hNedd4-WW4, placing it at the frontier of cooperative folding.

Isothermal Titration Calorimetry: Thermodynamic Analysis of the Binding Thermograms of Molecular Recognition Events by Using Equilibrium Models

© 2013 Martinez et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Isothermal Titration Calorimetry: Thermodynamic Analysis of the Binding Thermograms of Molecular Recognition Events by Using Equilibrium Models

Approaching the thermodynamic view of protein folding through the reproduction of Anfinsen's experiment by undergraduate physical biochemistry students

In 1972 Christian B. Anfinsen received the Nobel Prize in Chemistry for “…his work on ribonuclease, especially concerning the connection between the amino acid sequence and the biologically active conformation.” The understanding of this principle is crucial for physical biochemistry students, since protein folding studies, bio‐computing sciences and protein design approaches are founded on such a well‐demonstrated connection. Herein, we describe a detailed and easy‐to‐follow experiment to reproduce the most relevant assays carried out at Anfinsens laboratory in the 60s. This experiment provides students with a platform to interpret by themselves the structural and kinetic experiments conceived to understand the protein folding problem. In addition, this three‐day experiment brings students a nice opportunity for protein manipulation as well as for the setting up of spectroscopic and chromatographic techniques.

Chapter 12:Biocalorimetry: Differential Scanning Calorimetry of Protein Solutions

The term biocalorimetry refers to the application of calorimetry to the study of the energetics of biological processes. Current high-sensitivity commercially available calorimeters offer the possibility of using small sample volumes of very dilute solutions and are able to directly measure very small amounts of heat, which allow the analysis of the thermodynamics of non-covalent interactions, such as those involved in biological macromolecules. This chapter presents an overview of the most relevant applications of DSC to protein solution studies. A survey of the main theoretical models to interpret and analyze protein calorimetric thermograms is described, together with an evaluation of their applicability. Here the main goal of DSC is to characterize protein stability and analyze protein thermal unfolding. From the DSC measurements all the meaningful thermodynamic parameters of such processes, together with the temperature-dependent population of the initial, final and possible intermediate states can be determined. The wider possibilities of DSC to investigate the effect of protein self-association as well as that of the presence of protein ligands during the thermal processes are also described. Finally, when the unfolding process does not occur under equilibrium, DSC is also a very useful technique to study protein denaturation through a kinetic approach.