Van der Waals Interactions Dominate Ligand−Protein Association in a Protein Binding Site Occluded from Solvent Water

Abstract

In the present study we examine the enthalpy of binding of 2-methoxy-3-isobutylpyrazine (IBMP) to the mouse major urinary protein (MUP), using a combination of isothermal titration calorimetry (ITC), NMR, X-ray crystallography, all-atom molecular dynamics simulations, and site-directed mutagenesis. Global thermodynamics data derived from ITC indicate that binding is driven by favorable enthalpic contributions, rather than a classical entropy-driven signature that might be expected given that the binding pocket of MUP-1 is very hydrophobic. The only ligand-protein hydrogen bond is formed between the side-chain hydroxyl of Tyr120 and the ring nitrogen of the ligand in the wild-type protein. ITC measurements on the binding of IBMP to the Y120F mutant demonstrate a reduced enthalpy of binding, but nonetheless binding is still enthalpy dominated. A combination of solvent isotopic substitution ITC measurements and all-atom molecular dynamics simulations with explicit inclusion of solvent water suggests that solvation is not a major contributor to the overall binding enthalpy. Moreover, hydrogen/deuterium exchange measurements suggest that there is no significant contribution to the enthalpy of binding derived from "tightening" of the protein structure. Data are consistent with binding thermodynamics dominated by favorable dispersion interactions, arising from the inequality of solvent-solute dispersion interactions before complexation versus solute-solute dispersion interactions after complexation, by virtue of poor solvation of the binding pocket.

Water Sites, Networks, And Free Energies with Grand Canonical Monte Carlo

“…64 To investigate the effect of fixing the protein backbone on the water binding free energies, two sets of simulations were run, with four repeats each, of free and fixed protein backbone. The PDB structure 1QY1 was used to set-up the system before simulation.…”

“…A previous study, using two 9.5 ns molecular dynamics simulations and the TIP3P water model, estimated there to be between 3 and 4 waters bound in MUP-I cavity. 64 The optimum occupancy of a cavity can also be calculated without evaluating binding free energies by applying equation 6. Doing so on the unconstrained MUP-I simulations predicted an average The binding free energy of a given number of water molecules to MUP-I for simulations where the protein backbone was kept fixed, compared to when it was unconstrained.…”

High resolution X‐ray structures of mouse major urinary protein nasal isoform in complex with pheromones

“…24 z We note that all MUP structures, including those presented here, have been determined at pH 4.8-7.2. 21,23,24,[26][27][28][29][30][31][32] The pK a of the conjugate acid of thiazole and pyrazine derivatives is around pH 2, 38,39 thus, the sp2 hybridized nitrogen is less likely to be protonated at the pH of the experiments than is a Glu residue. To satisfy hydrogen bonding potential, we show E118 as protonated at OE2 in the figures.…”

“…22 X-ray and NMR structures of MUP-I and MUP-II have been determined in the presence of SBT 23,24 as well as other ligands. [24][25][26][27][28][29][30][31][32] However, until now, there have been no published structures of a group 4 isoform. We have determined the X-ray crystal structures of MUP-IV in complex with three mouse pheromones to examine the structural basis of ligand binding specificity.…”

Is there a link between selectivity and binding thermodynamics profiles?

“…Therefore, computational approaches can significantly facilitate the design of selective compounds, if high-quality crystal structures are available. Furthermore, the combination of experimental and computational approaches is able to rationalize unique cases where apolar contacts contribute to the favourable binding enthalpy, in a protein binding site occluded from solvent water [14].…”