posted on 2022-12-16, 13:06authored byQihui Liang, Yiying Zhang, Hong Zhang, Si Wu, Weibin Gong, Sarah Perrett
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 α-helical
lid, SBDα, 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 SBDα undergoes a conformational transition from
ordered to disordered after diamide stimulation. The disordered SBDα
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
SBDα 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.