Price for Opening the Transient Specificity Pocket in Human Aldose Reductase upon Ligand Binding: Structural, Thermodynamic, Kinetic, and Computational Analysis

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.