Serena G. Piticchio, Miriam Martínez-Cartró, Salvatore Scaffidi, Sergio Rodríguez-Arévalo,Andrea Bagán, Ainoa Sánchez-Arfelis, Sarah Picaud, Tobias Krojer, Panagis Filippakopoulos,Frank von Delft, Carmen Escolano, Carles Galdeano, and Xavier Barril

DOI:https://doi.org/10.1002/anie.202521138

The hydrophobic effect is a central force in molecular recognition, typically attributed to the ordering of watermolecules around apolar groups. Hydrophobic interaction sites on proteins are therefore readily predicted based on surfacepolarity. Yet, in the bromodomain-containing protein 4 (BRD4), a well-known hydrophobic hot spot is paradoxically linedby a network of water molecules. Here we combine binding assays, structural data, molecular dynamics, and free-energycalculations to resolve this apparent contradiction. We show that the water network functions as a hydrophobic recognitionmotif that cannot accommodate polar groups without disruption. Instead, as the protein pre-organizes the water network,apolar groups can bind with minimal entropic cost. In turn, they reinforce the surrounding hydrogen-bond network, limitingthe mobility of the entire protein–water assembly. With this perspective, we identify water networks potentially functioningas hydrophobic motifs in other pharmacological targets, revealing a general but overlooked recognition element with broadimplications in drug discovery and protein design