Thermodynamic and Structural Effects of Conformational Constraints in Protein−Ligand Interactions. Entropic Paradoxy Associated with Ligand Preorganization

Abstract

Succinate- and cyclopropane-derived phosphotyrosine (pY) replacements were incorporated into a series of Grb2 SH2 binding ligands wherein the pY+1 residue was varied to determine explicitly how variations in ligand preorganization affect binding energetics and structure. The complexes of these ligands with the Grb2 SH2 domain were examined in a series of thermodynamic and structural investigations using isothermal titration calorimetry and X-ray crystallography. The binding enthalpies for all ligands were favorable, and although binding entropies for all ligands having a hydrophobic residue at the pY+1 site were favorable, binding entropies for those having a hydrophilic residue at this site were unfavorable. Preorganized ligands generally bound with more favorable Gibbs energies than their flexible controls, but this increased affinity was the consequence of relatively more favorable binding enthalpies. Unexpectedly, binding entropies of the constrained ligands were uniformly disfavored relative to their flexible controls, demonstrating that the widely held belief that ligand preorganization should result in an entropic advantage is not necessarily true. Crystallographic studies of complexes of several flexible and constrained ligands having the same amino acid at the pY+1 position revealed extensive similarities, but there were some notable differences. There are a greater number of direct polar contacts in complexes of the constrained ligands that correlate qualitatively with their more favorable binding enthalpies and Gibbs energies. There are more single water-mediated contacts between the domain and the flexible ligand of each pair; although fixing water molecules at a protein-ligand interface is commonly viewed as entropically unfavorable, entropies for forming these complexes are favored relative to those of their constrained counterparts. Crystallographic b-factors in the complexes of constrained ligands are greater than those of their flexible counterparts, an observation that seems inconsistent with our finding that entropies for forming complexes of flexible ligands are relatively more favorable. This systematic study highlights the profound challenges and complexities associated with predicting how structural changes in a ligand will affect enthalpies, entropies, and structure in protein-ligand interactions.

Bridging Calorimetry and Simulation through Precise Calculations of Cucurbituril–Guest Binding Enthalpies

“…This is an important direction, because calorimetric data are often puzzling or even paradoxical. 62,16 For example, small changes in chemical structure often lead to correspondingly small changes in free energy, but seemingly disproportionately large changes in enthalpy and entropy. 8−13 Similarly, the efforts of medicinal chemists to enhance binding by making ligands more rigid, and thereby presumably reducing the entropy penalty, sometimes enhances the binding enthalpy instead of the entropy.…”

Entropy–enthalpy transduction caused by conformational shifts can obscure the forces driving protein–ligand binding

“…Moreover, information on changes in entropy and enthalpy can usefully guide the design of improved drug molecules (1), with advantageous specificity (2) and physical properties (3). However, calorimetric studies of biomolecular binding and folding often reveal unexpected changes in entropy and enthalpy that are difficult to interpret in terms of physical driving forces (4)(5)(6)(7)(8). Some of these puzzling results are instances of entropy-enthalpy compensation (9), a common but not universal (10,11) phenomenon in which perturbations of a system that increase the enthalpy also increase the entropy or vice versa; therefore, the net change in the free energy remains small.…”

Geometrically Constrained Derivatives of Indolylglyoxylamides as Ligands Binding the GABAA/BzR Complex

“…As the Gibbs energy comprises both enthalpic and entropic contributions, since G° = H° -T S°, optimization of the ligand-protein interaction should necessarily be obtained if the structure of the compound is modified so as to obtain both a more negative binding enthalpy and a more positive binding entropy. As a consequence, it may happen that constrained ligands, compared with their flexible parent compounds, bind target proteins with more favourable free energies, and accordingly a higher binding affinity, because they benefit from more favourable binding enthalpies, although displaying unfavourable entropies of binding [32][33][34][35][36].…”

“…Insertion of a carbonyl group within the side chain, to obtain arylalkylaminocarbonyl derivatives, led to similar results. Indeed, in both the (29-31) and the (32)(33)(34) series, an appreciable binding affinity was displayed only by compounds (29) (K i : 1.7 M) and (32) (K i : 1.6 M), unsubstituted on the side phenyl ring. Insertion of both an electronwithdrawing atom, like chloro, and an electron-releasing group, like OCH 3 , in the para position of their distal phenyl ring significantly reduced the activity.…”