{Reference Type}: Journal Article
{Title}: A finely balanced order-disorder equilibrium sculpts the folding-binding landscape of an antibiotic sequestering protein.
{Author}: Natarajan L;De Sciscio ML;Nardi AN;Sekhar A;Del Giudice A;D'Abramo M;Naganathan AN;
{Journal}: Proc Natl Acad Sci U S A
{Volume}: 121
{Issue}: 20
{Year}: 2024 May 14
{Factor}: 12.779
{DOI}: 10.1073/pnas.2318855121
{Abstract}: 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.