Intrinsically disordered regions (IDRs) are common and important functional domains in many proteins. However, IDRs are difficult to target for drug development due to the lack of defined structures that would facilitate the identification of possible drug-binding pockets. Galectin-3 is a carbohydrate-binding protein of which overexpression has been implicated in a wide variety of disorders, including cancer and inflammation. Apart from its carbohydrate-recognition/binding domain (CRD), Galectin-3 also contains a functionally important disordered N-terminal domain (NTD) that contacts the C-terminal domain (CTD) and could be a target for drug development. To overcome challenges involved in inhibitor design due to lack of structure and the highly dynamic nature of the NTD, we used a protocol combining nuclear magnetic resonance data from recombinant Galectin-3 with accelerated molecular dynamics (MD) simulations. This approach identified a pocket in the CTD with which the NTD makes frequent contact. In accordance with this model, mutation of residues L131 and L203 in this pocket caused loss of Galectin-3 agglutination ability, signifying the functional relevance of the cavity. In silico screening was used to design candidate inhibitory peptides targeting the newly discovered cavity, and experimental testing of only three of these yielded one peptide that inhibits the agglutination promoted by wild-type Galectin-3. NMR experiments further confirmed that this peptide indeed binds to a cavity in the CTD, not within the actual CRD. Our results show that it is possible to apply a combination of MD simulations and NMR experiments to precisely predict the binding interface of a disordered domain with a structured domain, and furthermore use this predicted interface for designing inhibitors. This procedure can potentially be extended to many other targets in which similar IDR interactions play a vital functional role.
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