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 ...

Well-ordered water molecules are displaced from thrombin's hydrophobic S3/4-pocket by P3-varied ligands (Gly, d-Ala, d-Val, d-Leu to d-Cha with increased hydrophobicity and steric requirement). Two series with 2-(aminomethyl)-5-chlorobenzylamide and 4-amidinobenzylamide at P1 were examined by ITC and crystallography. Although experiencing different interactions in S1, they display almost equal potency. For both scaffolds the terminal benzylsulfonyl substituent differs in binding, whereas the increasingly bulky P3-groups address S3/4 pocket similarly. Small substituents leave the solvation pattern unperturbed as found in the uncomplexed enzyme while increasingly larger ones stepwise displace the waters. Medium-sized groups show patterns with partially occupied waters. The overall 40-fold affinity enhancement correlates with water displacement and growing number of van der Waals contacts and is mainly attributed to favorable entropy. Both Gly derivatives deviate from the series and adopt different binding modes. Nonetheless, their thermodynamic signatures are virtually identical with the homologous d-Ala derivatives. Accordingly, unchanged thermodynamic profiles are no reliable indicator for conserved binding modes.

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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.

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