Abstract
The hydrophobic effect is a central force in molecular recognition, typically attributed to the ordering of water molecules around apolar groups. Hydrophobic interaction sites on proteins are therefore readily predicted based on surface polarity. Yet, in the bromodomain-containing protein 4 (BRD4), a well-known hydrophobic hot spot is paradoxically lined by a network of water molecules. Here we combine binding assays, structural data, molecular dynamics, and free-energy calculations to resolve this apparent contradiction. We show that the water network functions as a hydrophobic recognition motif 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, limiting the mobility of the entire protein-water assembly. With this perspective, we identify water networks potentially functioning as hydrophobic motifs in other pharmacological targets, revealing a general but overlooked recognition element with broad implications in drug discovery and protein design.