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

Abstract

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.

The Role of Displacing Confined Solvent in the Conformational Equilibrium of β-Cyclodextrin

“…In a recent joint experimental/computational study, Rechlin et al investigated the binding of a series of 2-carbamoyl-phenyloxy acetic acid derivatives to Human Aldose Reductase and found that the opening of a specific cleft in the binding pocket was dependent on ligand substituent due to protein reorganization and the ligand desolvation penalties. 69 MD simulations of the unbound protein found that the open conformation of this pocket was unstable relative to the closed conformation and solvation thermodynamic analysis confirmed the presence of energetically unfavorable water molecules inside the binding cleft. In another joint experimental and computational study, Maeno et al noted the existence of two states for the L99A mutant of T4 Lysozyme under high pressure conditions: a ground state with an enlarged hydrophobic cavity and a high energy state where a phenylalanine was flipped inside the cavity.…”

Which Properties Allow Ligands to Open and Bind to the Transient Binding Pocket of Human Aldose Reductase?

“…Yet, the inhibitors 3 and 4 as well as 5 and 6 are similar, especially with regard to their Gibbs free energy of binding. [4]. All measurements were performed in HEPES buffer pH 8.0 and are not corrected for overlaying protonation effects, thus only relative differences should be interpreted.…”

“…Remarkably, the carboxylate group at the acetic acid moiety of inhibitor 2 binds with only one well-defined orientation in the anion binding pocket. [4] in light green (A,B) and 2 (4QBX) [4] in dark green (C,D) bound to the active site of ALR-2. To distinguish the position of both conformations, b is highlighted by a slightly darker color.…”

Human aldose reductase unfolds through an intermediate

“…Extensive information is available in the literature about the structure and function of hAR, mainly related to active site of hAR from high-resolution crystal structures with a number of potential inhibitors 5 , flexibility in the hAR binding site pocket 6 and the thermodynamics of closing/opening of the specificity pocket within binding site pocket of hAR 7 . There is little investigation related to the folding/unfolding mechanism of hAR.…”