Abstract
The bromodomain and extraterminal domain (BET) family of proteins recognizes diverse peptide motifs via their conserved ET domains, yet exhibits paralog-specific binding preferences with unclear structural origins. Here, we use extensive molecular dynamics simulations and ensemble-based analyses to investigate how two known peptide epitope binders - one from the host regulatory protein NSD3 and one from the murine leukemia virus integrase - interact with the ET domains of BRD3 and BRD4. Our results reveal that differences in peptide recognition are not driven by large conformational changes, but by subtle variations in loop dynamics and local secondary structure. Two divergent residues (positions 35 and 36) in the flexible loop connecting helices α2 and α3 shape the conformational ensemble of each paralog by modulating the formation of flanking helices ( η1 and η2 ), which in turn control the opening of the peptide-binding cavity. These intrinsic dynamical differences influence the number and stability of accessible binding modes, with BRD3 showing greater plasticity and weaker binding, particularly for NSD3. Our findings support a model in which local sequence variation tunes conformational selection and induced fit mechanisms, offering a structural rationale for paralog-specific targeting of BET proteins.