Mechanism of the hydrophobic effect in the biomolecular recognition of arylsulfonamides by carbonic anhydrase

Abstract

The hydrophobic effect-a rationalization of the insolubility of nonpolar molecules in water-is centrally important to biomolecular recognition. Despite extensive research devoted to the hydrophobic effect, its molecular mechanisms remain controversial, and there are still no reliably predictive models for its role in proteinligand binding. Here we describe a particularly well-defined system of protein and ligands-carbonic anhydrase and a series of structurally homologous heterocyclic aromatic sulfonamides-that we use to characterize hydrophobic interactions thermodynamically and structurally. In binding to this structurally rigid protein, a set of ligands (also defined to be structurally rigid) shows the expected gain in binding free energy as hydrophobic surface area is added. Isothermal titration calorimetry demonstrates that enthalpy determines these increases in binding affinity, and that changes in the heat capacity of binding are negative. X-ray crystallography and molecular dynamics simulations are compatible with the proposal that the differences in binding between the homologous ligands stem from changes in the number and organization of water molecules localized in the active site in the bound complexes, rather than (or perhaps in addition to) release of structured water from the apposed hydrophobic surfaces. These results support the hypothesis that structured water molecules-including both the molecules of water displaced by the ligands and those reorganized upon ligand binding-determine the thermodynamics of binding of these ligands at the active site of the protein. Hydrophobic effects in various contexts have different structural and thermodynamic origins, although all may be manifestations of the differences in characteristics of bulk water and water close to hydrophobic surfaces.physical-organic | entropy | surface water | benzo-extension | hydration T he hydrophobic effect-the energetically favorable association of nonpolar surfaces in an aqueous solution-often dominates the free energy of binding of proteins and ligands (1-5). Frequently, increasing the nonpolar surface area of a ligand decreases its dissociation constant (K d ; i.e., increases the strength of binding) (6), and simultaneously decreases its equilibrium constant for partitioning from a hydrophobic phase to aqueous solution (K P ) (7). Modern, structure-guided, ligand design has relied upon the "lock-and-key" notion of conformal association between the atoms of the ligand and the binding pocket of a protein; the detailed molecular basis for the hydrophobic effect, however, continues to be poorly understood (1-5). This lack of understanding of the hydrophobic effect prevents accurate prediction of the free energy of binding of proteins and ligands.The first, and currently most pervasive, rationale for the hydrophobic effect was based on studies of the thermodynamics of partitioning of nonpolar solutes from hydrophobic phases (i.e., the gas phase or a hydrophobic liquid phase) into water. The thermodynamics of partitioning of solut...

Water‐Restructuring Mutations Can Reverse the Thermodynamic Signature of Ligand Binding to Human Carbonic Anhydrase

“…The additional benzo ring of BTA increases its binding affinity (relative to TA) through an enthalpically favorable hydrophobic interaction with the nonpolar wall of HCAII. [17] By examining the binding of TA and BTA to mutants of HCAII (with mutations localized in the binding pocket), we sought to establish the thermodynamic influence of mutations on protein-ligand association in this system. A comparison of binding between these two ligands, in turn, enabled us to examine how mutations affect the thermodynamics of hydrophobic association.…”

“…A comparison of binding between these two ligands, in turn, enabled us to examine how mutations affect the thermodynamics of hydrophobic association. (This study relies on the "benzo-extension" strategy, which we have described and exploited previously [17] ).…”

“…[11,12,17,29,30] Hydrophobic interactions associated with large, favorable changes in entropy, for example, might involve the expulsion of well-ordered molecules of water from the binding pocket, while those associated with large, favorable changes in enthalpy might result from the expulsion and/or reorganization of poorly-ordered water. To examine the influence of mutations on the thermodynamic signature of the hydrophobic effect in our system, we calculated the difference in thermodynamic binding parameters for BTA and TA (i.e., ΔΔJ°b -benzo = ΔJ°b -BTA -ΔJ°b -TA ), a difference associated with the hydrophobic interaction between the benzo ring of BTA and the nonpolar wall of HCAII.…”

Is there a link between selectivity and binding thermodynamics profiles?

“…Saquinavir and TMC-126 have the same number of polar groups, however Saquinavir binding is associated with unfavourable ~+5 kJ/mol binding enthalpy while TMC-126 binding is significantly more enthalpic (H~-50 kJ/mol) due to the better orientation of its polar groups [7]. It should also be noted that binding-site water molecules have complex influence on thermodynamics signatures [8][9][10]. Orientation of polar groups largely influences their specific interactions compared to non-polar functional groups that are introduced to fill apolar cavities.…”

“…Replacement of unstable water molecules within hydrophobic pockets is mostly driven by entropy changes, although enthalpy gain coupled with water replacement by apolar moieties had also been reported [8][9]. The effect of binding site waters has been recently reviewed by using WaterMap for solvation energetic calculations [11].…”