Abstract
Molecular recognition is fundamental to biological signaling. A central question is how individual interactions between molecular moieties affect the thermodynamics of ligand binding to proteins and how these effects might propagate beyond the immediate neighborhood of the binding site. Here, we investigate this question by introducing minor changes in ligand structure and characterizing the effects of these on ligand affinity to the carbohydrate recognition domain of galectin-3, using a combination of isothermal titration calorimetry, X-ray crystallography, NMR relaxation, and computational approaches including molecular dynamics (MD) simulations and grid inhomogeneous solvation theory (GIST). We studied a congeneric series of ligands with a fluorophenyl-triazole moiety, where the fluorine substituent varies between the ortho, meta, and para positions (denoted O, M, and P). The M and P ligands have similar affinities, whereas the O ligand has 3-fold lower affinity, reflecting differences in binding enthalpy and entropy. The results reveal surprising differences in conformational and solvation entropy among the three complexes. NMR backbone order parameters show that the O-bound protein has reduced conformational entropy compared to the M and P complexes. By contrast, the bound ligand is more flexible in the O complex, as determined by 19F NMR relaxation, ensemble-refined X-ray diffraction data, and MD simulations. Furthermore, GIST calculations indicate that the O-bound complex has less unfavorable solvation entropy compared to the other two complexes. Thus, the results indicate compensatory effects from ligand conformational entropy and water entropy, on the one hand, and protein conformational entropy, on the other hand. Taken together, these different contributions amount to entropy–entropy compensation among the system components involved in ligand binding to a target protein.
Highlights
Binding of low-molecular-weight ligands to proteins is fundamental to a large number of signaling pathways in biology and of central interest in drug design aiming to interfere with such pathways for medicinal purposes
We identified water sites and analyzed the thermodynamics of the solvent around the galectin-3C complexes, using grid inhomogeneous solvation theory (GIST),[76,77] implemented in the cpptraj module of the Amber software
The isothermal titration calorimetry (ITC) experiments reveal that the complexes have similar thermodynamic signatures overall, which might be expected from the similar structure of the ligands
Summary
Binding of low-molecular-weight ligands to proteins is fundamental to a large number of signaling pathways in biology and of central interest in drug design aiming to interfere with such pathways for medicinal purposes. The main challenge involves the fact that the free energy of binding is manifested as a small difference between large numbers originating from a vast number of interactions between the protein, ligand, other solutes, and solvent molecules. Changes in conformational fluctuations in proteins upon ligand binding can give rise to significant entropic contributions to affinity.[1−8] Ligand binding may affect the water network in and around binding sites, causing appreciable effects on the thermodynamics of solvation.[8−13]. Article solvation in ligand binding.[8,13] Galectin-3 is an interesting model system in this regard, because its binding site is relatively solvent-accessible, being located in a shallow groove across one of the two β-sheets. Several water molecules form an integral part of the ligand environment by forming bridging hydrogen bonds between the ligand and protein.[13,14] Galectin-
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