Structural Superimposition

Abstact

Enthalpy-entropy compensation is a widely observed feature of protein-ligand recognition, yet its structural origins remain difficult to resolve. Here, we investigate a matched series of sulfonamide inhibitors binding to three closely related human carbonic anhydrase (CA) isozymes (CA II, CA XII, and CA XIII) using isothermal titration calorimetry, fluorescence thermal shift assays, and high-resolution X-ray crystallography. Although these isozymes share nearly identical overall folds and exhibit highly similar intrinsic Gibbs energies of binding, their enthalpic and entropic contributions diverge markedly. Correction of all observed thermodynamic quantities for proton-linked equilibria reveals that this divergence originates from subtle differences in the active-site microenvironment. In particular, single-residue variations-including the Thr/Thr/Val substitution at position 200, the Phe/Ala/Phe variation at position 131, and the Asn/Asn/Ser difference at position 62-modulate hydrogen-bond geometry, steric complementarity, and local solvation, thereby reshaping the balance between enthalpic and entropic driving forces. These results demonstrate how closely related proteins can achieve similar binding affinities through distinct energetic strategies and establish a coherent structure-thermodynamics framework for sulfonamide recognition.