Deciphering the molecular mechanisms underlying multi-drug resistance in bacteria is crucial for developing novel therapeutics. One such mechanism of antibiotic resistance is their sequestration via protein-based sensors. TipAS and AlbAS, synthesized by Streptomyces lividans and Klebsiella oxytoca, respectively, bind and sequester antibiotics. TipAS sequesters multiple thiopeptides in its partially disordered N-terminal sub-domain that folds upon binding, while AlbAS that harbors two TipAS-like effector binding domains binds albicidin in a central tunnel that spans the protein. The precise extent and role of protein dynamics in enabling promiscuous binding in otherwise occluded binding sites are open questions. Combining equilibrium and time-resolved spectroscopy with calorimetry and statistical modeling, we find that the TipAS native ensemble exhibits a pre-equilibrium between binding-incompetent and competent substates. Binding to thiostrepton follows a combination of induced-fit and conformational-selection-like mechanisms, via partial binding and concomitant stabilization of the binding-competent substate. These ensemble features are evolutionarily conserved across orthologs from select bacteria, underscoring the critical role of disorder in the native ensemble of antibiotic-sequestering MerR proteins. Tryptophan-scanning mutagenesis, kinetics, and HDX-MS analysis of AlbAS unfolding showcases the presence of pervasive dynamics across the entire protein illustrating a possible mechanism for albicidin sequestration. Our work thus highlights how MerR family effector binding domains sequester ligands through breathing motions that involves not just local unfolding but also large-scale order-disorder transitions.