Reversible Redox-Dependent Conformational Switch of the C-Terminal α-Helical Lid of Human Hsp70 Observed by In-Cell NMR

Glutathionylation of human stress-inducible Hsp70 (hHsp70) under oxidative stress conditions has been suggested to act as an on/off switch of hHsp70 chaperone activity and thus transfer redox signals to hHsp70 clients through a change in conformation. The mechanism of this switch involves unfolding of the C-terminal alpha-helical lid, SBD alpha, upon glutathionylation, which then binds to and blocks the hHsp70 substrate-binding site. This process is reversible and redox-regulated and has been demonstrated for purified protein in solution. Here, we found that this redox-regulated reversible process also occurs in the cellular environment. Using Escherichia coli as a model system, in -cell NMR data clearly indicate that hHsp70 SBD alpha undergoes a conformational transition from ordered to disordered after diamide stimulation. The disordered SBD alpha could spontaneously recover back to the helix bundle conformation over time. This oxidative-stress induced process also occurred in cell lysate, with a similar unfolding rate as in cells, but the refolding rate was significantly slower in cell lysate. Increased temperature accelerates this process. Under heat stress alone, unfolding of the SBD alpha could not be detected in cells. Our in-cell NMR results provide direct support for the molecular switch model of hHsp70 redox regulation and also demonstrate the power of in-cell NMR for real-time study of protein structures during biological processes in living cells.