Structural basis and mechanism of the unfolding-induced activation of HdeA, a bacterial acid response chaperone.

The role of protein structural disorder in biological functions has gained increasing attention in the past decade. The bacterial acid-resistant chaperone HdeA belongs to a group of "conditionally disordered" proteins, because it is inactive in its well-structured state and becomes activated via an order-to-disorder transition under acid stress. However, the mechanism for unfolding-induced activation remains unclear because of a lack of experimental information on the unfolded state conformation and the chaperone-client interactions. Herein, we used advanced solution NMR methods to characterize the activated-state conformation of HdeA under acidic conditions and identify its client-binding sites. We observed that the structure of activated HdeA becomes largely disordered and exposes two hydrophobic patches essential for client interactions. Furthermore, using the pH-dependent chemical exchange saturation transfer (CEST) NMR method, we identified three acid-sensitive regions that act as structural locks in regulating the exposure of the two client-binding sites during the activation process, revealing a multistep activation mechanism of HdeA's chaperone function at the atomic level. Our results highlight the role of intrinsic protein disorder in chaperone function and the self-inhibitory role of ordered structures under nonstress conditions, offering new insights for improving our understanding of protein structure-function paradigms.