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The binding of sulfonamides to human carbonic anhydrase II (hCAII) is a complex and long-debated example of protein-ligand recognition and interaction. In this study, we investigate the para-substituted n-alkyl and hydroxyethylene-benzenesulfonamides, providing a complete reconstruction of their binding pathway to hCAII by means of large-scale molecular dynamics simulations, density functional calculations, surface plasmon resonance (SPR) measurements, and X-ray crystallography experiments. Our analysis shows that the protein-ligand association rate (kon) dramatically increases with the ligand's hydrophobicity, pointing to the existence of a prebinding stage largely stabilized by a favorable packing of the ligand's apolar moieties with the hCAII "hydrophobic wall". The characterization of the binding pathway allows an unprecedented understanding of the structure-kinetic relationship in hCAII/benzenesulfonamide complexes, depicting a paradigmatic scenario for the multistep binding process in protein-ligand systems. [ABSTRACT FROM AUTHOR]

Insights into the thermodynamic and kinetic signature of the transient opening of a protein-binding pocket resulting from accommodation of suitable substituents attached to a given parent ligand scaffold are presented. As a target, we selected human aldose reductase, an enzyme involved in the development of late-stage diabetic complications. To recognize a large scope of substrate molecules, this reductase opens a transient specificity pocket. The pocket-opening step was studied by X-ray crystallography, microcalorimetry, and surface plasmon resonance using a narrow series of 2-carbamoyl-phenoxy-acetic acid derivatives. Molecular dynamics simulations suggest that pocket opening occurs only once an appropriate substituent is attached to the parent scaffold. Transient pocket opening of the uncomplexed protein is hardly recorded. Hydration-site analysis suggests that up to five water molecules entering the opened pocket cannot stabilize this state. Sole substitution with a benzyl group stabilizes the opened state, and the energetic barrier for opening is estimated to be ∼5 kJ/mol. Additional decoration of the pocket-opening benzyl substituent with a nitro group results in a huge enthalpy-driven potency increase; on the other hand, an isosteric carboxylic acid group reduces the potency 1000-fold, and binding occurs without pocket opening. We suggest a ligand induced-fit mechanism for the pocket-opening step, which, however, does not represent the rate-determining step in binding kinetics.