Abstract
Proteins frequently undergo large-scale conformational excursions in their native ensemble. Such structural transitions are particularly critical for enabling access to binding sites when they are buried in the protein interior. Here, we map the conformational landscape of AlbAS, a natural isoform of the transcription factor AlbA from the gut microbe Klebsiella oxytoca, which sequesters the antibiotic albicidin in a solvent-inaccessible binding tunnel. Combining equilibrium, time-resolved experiments, structural mass spectrometry and calorimetry with statistical modeling, we show that AlbAS displays large differences in local and global stability and dynamics, with ∼600-fold difference in unfolding rates across different parts of the structure. Several residues lining the ligand-binding pocket and the inter-sub-domain residues rapidly exchange protons with the solvent in hydrogen-deuterium exchange mass spectrometry experiments, indicative of anisotropic distributions of local stabilities, with the N-terminal subdomain being less stable. The AlbAS conformational landscape is thus quite rugged, encompassing numerous partially structured states in equilibrium, including partial unlocking of the N-terminal subdomain at a time-constant of 6 ms that exposes the binding sites to aid in albicidin binding.
Keywords:
antibiotic resistance; antibiotic sequestration; domain duplication; protein dynamics; tryptophan scanning mutagenesis.