A finely balanced order–disorder equilibrium sculpts the folding–binding landscape of an antibiotic sequestering protein

TipA, a MerR family transcription factor from Streptomyces lividans , promotes antibiotic resistance by sequestering broad - spectrum thiopeptide - based antibiotics, thus counteracting their inhibitory effect on ribosomes. TipAS, a minimal binding motif which is expressed as an isoform of TipA, harbors a partially disordered N - terminal subdomain that folds upon binding multiple antibiotics. The extent and nature of the underlying molecular heterogeneity in TipAS that shapes its promiscuous folding-function landscape is an open question and is critical for understanding antibiotic - sequestration mechanisms. Here, combining equilibrium and time - resolved experiments, statistical modeling, and simulations, we show that the TipAS native ensemble exhibits a pre - equilibrium between binding - incompetent and binding - competent substates, with the fully folded state appearing only as an excited state under physiological conditions. The binding - competent state characterized by a partially structured N - terminal subdomain loses structure progressively in the physiological range of temperatures, swells on temperature increase, and displays slow conformational exchange across multiple conformations. Binding to the bactericidal antibiotic 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 that infect humans, underscoring the functional role of partial disorder in the native ensemble of antibiotic - sequestering proteins belonging to the MerR family.