¹⁹F NMR Probe The Chaperone HdeA Acid Activation Mechanism

The extremely acidic environment (pH 1-3) of mammal stomach not only serves to promote food digestion, but also acts as a natural barrier against extrinsic microbes. Enterohemorrhagic E. coli can break through this host defense and cause diseases. Recently research has reported there is an acid chaperone HdeA in E. coli periplasm, which is an inactive, structured dimer at neutral pH. Once in the stomach HdeA dissociates into an active partially unfolded monomer, binds denatured substrates and prevents their aggregation. Although X-ray crystallography had solved the 3D structure of HdeA as a well-folded dimer at neutral pH, detailed structural information about the active monomer is sparse. How about the dynamics and the mode of substrate binding? Do some parts remain structured while others are unstructured? What is the role of the intramolecular disulfide bond? These important issues still await to answer.19F NMR is a powerful tool for studying dynamics and interactions of biomolecular, because of its high sensitivity, spectral simplicity and large chemical shift range. Here we first apply 19F as NMR probe into chaperone system.19F NMR provides quantitative insight into HdeA unfolding and activation, and unfolding is necessary but insufficient for activation. At acid pH HdeA is conformationally heterogeneous and the multiple conformations are in dynamic exchange on chemical shift timescale. Between the two dominant conformations, only the partially folded form can bind substrates, while the necessary existence of the unfolded form is suspected to protect or support the active form. Only the disulfide region shows residual structure and others all are disordered. The disulfide is the structural key to maintain the partially folded active form and also provides the structural basis for refolding. Our results perfectly answer the questions above.Chaperone HdeA is a typical conditionally disordered protein, which can be an attractive protein model for structural biologists. Our results suggest that partial folding is essential for chaperone activation. Once the local residual structure is lost, the completed unfolded structure leads to the loss of activity. Order and disorder cooperatively control HdeA function. This new structural mechanism increases our mechanistic understanding of other chaperones and may lead to improved treatments for bacterial diseases. The ability to detect multiple conformations and their exchange dynamics shows that 19F NMR is a powerful tool and can be well applied to study other chaperones.