Targeting interactions between the Galectin-3 intrinsically disordered and structured domains based on long time-scale accelerated molecular dynamics

Abstract

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 which 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 novel protocol combining nuclear magnetic resonance data from recombinant Galectin-3 with accelerated molecular dynamics (MD) simulations to identify a shallow pocket in the CTD with which the NTD makes frequent contact. In accordance with this model, a Galectin-3 double mutant of residues L131 and L203 in the CTD lost agglutination ability. In-silico design was used to narrow down candidate inhibitory peptides and experimental testing of only 3 of these yielded one peptide that inhibits the agglutination promoted by wild type Galectin-3. NMR experiments further confirmed that this peptide makes contacts with a non-carbohydrate binding moiety of the CTD. 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 thus be potentially extended to many other targets in which similar IDR interactions play a vital functional role.