Abstract
AbstractThis study uses mutants of human carbonic anhydrase (HCAII) to examine how changes in the organization of water within a binding pocket can alter the thermodynamics of protein–ligand association. Results from calorimetric, crystallographic, and theoretical analyses suggest that most mutations strengthen networks of water‐mediated hydrogen bonds and reduce binding affinity by increasing the enthalpic cost and, to a lesser extent, the entropic benefit of rearranging those networks during binding. The organization of water within a binding pocket can thus determine whether the hydrophobic interactions in which it engages are enthalpy‐driven or entropy‐driven. Our findings highlight a possible asymmetry in protein–ligand association by suggesting that, within the confines of the binding pocket of HCAII, binding events associated with enthalpically favorable rearrangements of water are stronger than those associated with entropically favorable ones.
Highlights
Biomolecular recognition is a process influenced as much by rearrangements in the molecules of water that solvate interacting species as it is by the interactions between those species.[1,2,3]
We examined the influence of amino acid substitutions on the thermodynamics of protein-ligand association by using isothermal titration calorimetry (ITC) to determine the enthalpy, entropy, and free energy of binding (ΔH°b, -TΔS°b, and ΔG°b) for each combination of ligand and mutant
To examine how mutations might alter the thermodynamic properties of water in the binding pocket, we used (i) X-ray crystallography to collect crystal structures of a subset of mutant-BTA complexes and (ii) WaterMap (Schrödinger Inc.[22,23,24]) to calculate the enthalpy and entropy of water—that is, the change in enthalpy and entropy associated with the transfer of a molecule of water from the bulk—in crystallographically determined binding pockets with and without BTA bound.[25,26]
Summary
Biomolecular recognition is a process influenced as much by rearrangements in the molecules of water that solvate interacting species as it is by the interactions between those species.[1,2,3] A detailed understanding of the mechanisms by which these rearrangements contribute to the thermodynamics of association between solutes is, essential for predicting (and manipulating) the energetics of binding in biological systems.[4,5,6]. We examined the influence of amino acid substitutions on the thermodynamics of protein-ligand association by using ITC to determine the enthalpy, entropy, and free energy of binding (ΔH°b, -TΔS°b, and ΔG°b) for each combination of ligand and mutant (see Table S2 of Supporting Information).
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