Conformational Entropy and Protein Flexibility in Drug Design Studied by NMR Spectroscopy

Abstract: Proteins are complex molecules, present in all of the vital functions of life. The function of a protein is regulated by interactions between protein and other molecules. Drug design in pharmaceutical science aims to regulate the function of a protein by the design of synthesized molecules that binds to a protein with high affinity. A change in protein flexibility upon ligand binding can provide significant effects on the affinity of the ligand, where the change in flexibility can be related to entropy, a fundamental thermodynamic parameter. The focus of this thesis has been the characterization of ligand-induced changes in conformational entropy using nuclear magnetic resonance (NMR) spectroscopy experiments in combination with other techniques. NMR relaxation experiments and molecular dynamics (MD) simulations were used to characterize changes in the dynamics of backbone and side chain amides in the carbohydrate recognition domain of Galectin-3 (Gal3) upon binding of lactose. Order parameters determined from NMR relaxation experiments and MD simulations for backbone amides agreed qualitatively and showed an increased flexibility upon binding lactose, thus giving a significant and favourable contribution from conformational entropy to the free energy of binding. Using a combination of NMR spectroscopy, isothermal titration calorimetry (ITC), and X-ray crystallography, we characterized the binding of three ligands to Gal3, where the dissociation constants ranged over 2 orders of magnitude. 15N and 2H spin relaxation measurements showed that protein backbone and side chains respond to ligand binding by, on average, increased conformational fluctuations, where the response in increased fluctuations differs for the three ligands. The results reveal an intricate interplay between structure and conformational fluctuations in the ligand-bound complexes that fine-tunes the affinity. The estimated change in conformational entropy is comparable in magnitude to the binding enthalpy, demonstrating that it contributes favorably and significantly to ligand binding. The binding of matrix metalloprotease 12 (MMP12) was dissected using two enantiomeric inhibitors, RR and SS, with dissociation constants differing by one order of magnitude. Enantiomers have identical physical properties and chemical potentials in solvent. Thus, any differences between two enantiomers in their binding thermodynamics must be due to properties in the bound states. The inhibitors represent a novel class of MMP12 inhibitors, called hydroxy-hydantoins that comprise a weak zinc-binding group, a hydantoin, and a lipophilic biphenyl moiety, which is connected to the hydantoin via a carbinol linker. The binding was characterized using a combination of NMR spectroscopy, ITC, X-ray spectroscopy and MD-simulations. ITC shows highly separate thermodynamic patterns for the two enantiomers upon binding, due to a 180 degree flip of the zinc-coordinating hydantoin ring between RR- and SS-MMP12. The binding of the stronger inhibitor RR is driven by a favourable enthalpic contribution to the free energy of binding, whereas the binding of the weaker inhibitor SS is driven by an entropic contribution. NMR relaxation experiments and MD simulations indicate a favourable contribution to the free energy of binding from the change in protein flexibility upon binding the stronger inhibitor RR in comparison to the weaker inhibitor SS, which is supported by thermodynamic integration using MD simulations. Thus, the change in conformational entropy for binding RR is favourable, whereas the total change in entropy for binding is more favourable for binding SS, yielding conflicting results regarding the entropic contributions to the free energy of binding. The binding process of Gal3 was characterized using CPMG relaxation dispersion experiments for 3 different ligands. The apo-state of Gal3 exhibits intrinsic conformational exchange, where a minor population samples the bound state. The bound-states of Gal3 exhibited conformational exchange, where fitted chemical shifts from relaxation data correlates with chemical shift differences between ligand- and apo-states. Off-rates for the ligands correlate with the dissociation constants of the ligands, thus indicating that on-rates are identical and diffusion controlled for all ligands. The off-rates for the designed ligands are significantly slower, demonstrating that these stabilize the bound conformation, but do not affect the transition barrier of ligand binding. A 3-state binding process is proposed, where apo-Gal3 is present in a non-binding ground-state which samples a binding competent high-energy state.