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Dedicated to Professor Jack D. Dunitz on the occasion of his 90th birthdayThe hydrophobic effect is viewed as the driving force for the aggregation of nonpolar substances with extended lipophilic molecular surfaces in aqueous solution through the exclusion of water molecules from the formed interfaces. [1,2] It is usually quoted to explain why an oil/water mixture spontaneously separates, why soluble proteins fold with a hydrophobic core and a hydrophilic outer surface, [3,4] why membrane components assemble as lipid bilayers and micelles, why membrane proteins are accommodated in membrane segments, and why small molecules associate in protein binding pockets with mutual burial of hydrophobic surfaces. [5] In the latter instance, it is a general strategy in medicinal chemistry to improve protein-ligand binding by increasing the ligands hydrophobic surface which becomes buried in hydrophobic pockets of the target protein. In all cases, the hydrophobic effect is considered to be the major force of association. On the molecular level, this phenomenon is commonly attributed to the displacement of water molecules arranged around the hydrophobic surfaces, and entropic effects are made responsible to drive this association. The entropic profile is related to changes in the degree of ordering and the dynamic properties of the water molecules, which are assumed to be more disordered in the bulk water phase relative to where they were located prior to being displaced upon hydrophobic association. Recent studies have demonstrated, however, that hydrophobic interactions can originate either from enthalpyor entropy-driven binding, making simple explanations often presented for the hydrophobic effect insufficient. [6][7][8][9][10][11][12] Also in computational design tools the handling of explicit water molecules has received increasing recognition. Tools such as WaterMap and Szmap [13,14] try to take into account water structures in drug design and the properties of individual water molecules are discussed in terms of enthalpy and entropy.In order to obtain a better understanding of the hydrophobic effect on the molecular level and its role in proteinligand binding, we embarked on a systematic study using thermolysin (TLN) as a model system. [6] This thermostable bacterial zinc metalloprotease from Bacillus thermoproteolyticus exhibits three specificity pockets of predominantly hydrophobic nature (Scheme 1). It has been considered a prototype for the entire class of enzymes [15] owing to its highly conserved active-site architecture, despite remarkable sequence differences to other zinc proteases. Potent TLN inhibitors are often designed as transition-state analogues. [16][17][18] The enzyme has been frequently used as a surrogate [19][20][21][22][23] for other metalloenzymes against which new drugs are developed, and served as a model system to test ideas [24,25] and new methodological concepts. [26] TLN was one of the first crystallographically investigated metalloproteases [27,28] and its catalytic zinc ion is coordinated ...

Water is ubiquitously present in any biological system and has therefore to be regarded as an additional binding partner in the protein-ligand binding process. Upon complex formation, a new solvent-exposed surface is generated and water molecules from the first solvation layer will arrange around this newly formed surface. So far, the influence of such water arrangements on the ligand binding properties is unknown. In this study, the binding modes of nine congeneric phosphonamidate-type inhibitors with systematically varied, size-increasing hydrophobic P2 ' substituents (from methyl to phenylethyl) addressing the hydrophobic, solvent-exposed S2 ' pocket of thermolysin were analyzed by high-resolution crystal structures and correlated with their thermodynamic binding profiles as measured by isothermal titration calorimetry. Overall, ΔΔG spreads over 7.0 kJ mol(-1) , ΔΔH varies by 15.8 kJ mol(-1) , and -TΔΔS by 12.1 kJ mol(-1) . Throughout the series, these changes correlate remarkably well with the geometric differences of water molecules arranged adjacent to the P2 ' substituents. Ligands with medium-sized P2 ' substituents exhibit highest affinities, presumably because of their optimal solvation patterns around these complexes. The addition, removal, or rearrangement of even a single methyl group can result in a strong modulation of the adjacent water network pattern shifting from enthalpy to entropy-driven binding. In conclusion, the quality of a water network assembled around a protein-ligand complex influences the enthalpy/entropy signature and can even modulate affinity to a surprising extent.

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The binding of four congeneric peptide-like thermolysin inhibitors has been studied by high-resolution crystal structure analysis and isothermal titration calorimetry. The ligands differ only by a terminal carboxylate and/or methyl group. A surprising non-additivity of functional group contributions for the carboxylate and/or methyl groups is detected. Adding the methyl first and then the carboxylate group results in a small Gibbs free energy increase and minor enthalpy/entropy partitioning for the first modification, whereas the second involves a strong affinity increase combined with large enthalpy/entropy changes. However, first adding the carboxylate and then the methyl group yields reverse effects: the acidic group attachment now causes minor effects, whereas the added methyl group provokes large changes. As all crystal structures show virtually identical binding modes, affinity changes are related to rearrangements of the first solvation layer next to the S(2)' pocket. About 20-25 water molecules are visible next to the studied complexes. The added COO(-) groups perturb the local water network in both carboxylated complexes, and the attached methyl groups provide favorable interaction sites for water molecules. Apart from one example, a contiguously connected water network between protein and ligand functional groups is observed in all complexes. In the complex with the carboxylated ligand, which still lacks the terminal methyl group, the water network is unfavorably ruptured. This results in a surprising thermodynamic signature showing only a minor affinity increase upon COO(-) group attachment. Because the further added methyl group provides a favorable interaction site for water, the network can be reestablished, and a strong affinity increase with a large enthalpy/entropy signature is then detected.