Unfolding of Metastable Linker Region Is at the Core of Hsp33 Activation as a Redox-regulated Chaperone

Abstract

Hsp33, a molecular chaperone specifically activated by oxidative stress conditions that lead to protein unfolding, protects cells against oxidative protein aggregation. Stress sensing in Hsp33 occurs via its C-terminal redox switch domain, which consists of a zinc center that responds to the presence of oxidants and an adjacent metastable linker region, which responds to unfolding conditions. Here we show that single mutations in the N terminus of Hsp33 are sufficient to either partially (Hsp33-M172S) or completely (Hsp33-Y12E) abolish this posttranslational regulation of Hsp33 chaperone function. Both mutations appear to work predominantly via the destabilization of the Hsp33 linker region without affecting zinc coordination, redox sensitivity, or substrate binding of Hsp33. We found that the M172S substitution causes moderate destabilization of the Hsp33 linker region, which seems sufficient to convert the redox-regulated Hsp33 into a temperature-controlled chaperone. The Y12E mutation leads to the constitutive unfolding of the Hsp33 linker region thereby turning Hsp33 into a constitutively active chaperone. These results demonstrate that the redox-controlled unfolding of the Hsp33 linker region plays the central role in the activation process of Hsp33. The zinc center of Hsp33 appears to act as the redox-sensitive toggle that adjusts the thermostability of the linker region to the cell redox status. In vivo studies confirmed that even mild overexpression of the Hsp33-Y12E mutant protein inhibits bacterial growth, providing important evidence that the tight functional regulation of Hsp33 chaperone activity plays a vital role in bacterial survival.

Protein Quality Control under Oxidative Stress Conditions

“…Once exposed to the appropriate oxidative stress conditions, the four cysteines engage in two disulfide bonds, zinc is released and the zinc-binding domain unfolds. What is truly crucial for the activation of Hsp33, however, is the unfolding of the central linker region (99), which connects Hsp33’s N-terminus with the C-terminal redox switch domain. Not particularly conserved but highly charged, this linker region is stably folded under reducing, non-stress conditions yet rapidly unfolds upon the formation of both disulfide bonds.…”

Hsp33 Controls Elongation Factor-Tu Stability and Allows Escherichia coli Growth in the Absence of the Major DnaK and Trigger Factor Chaperones

“…The PCR fragment was digested with EcoRI and HindIII and cloned into either pSE380⌬NcoI or p29SEN previously digested with the same enzymes. A similar cloning procedure was used to construct the pSE-Hsp33(Y12E), pSE-Hsp33(M172S) mutant derivatives, except that in this case, plasmids pET11a-hslO(Y12E) or pET11a-hslO(M172S) DNA were used as a template (27). Plasmid pSE-Hsp33-FLAG (pSE380⌬NcoI-Hsp33-flag tagged) containing Hsp33 with the C-terminal FLAG tag "GSDYKD-DDDKSA" was constructed using primers Hsp33-for and 33FT-rev (5Ј-GCAAGCTTGGATCCTTAGGCGCTTTTAT-CGTCGTCATCTTTGTAGTCGCTGCCATGAACTTGCG-GATCTGC-3Ј) and pSE-Hsp33 as DNA template.…”

“…Instead, these two mutant Hsp33 proteins co-purified with the protein aggregates. Although it is known that Hsp33(Y12E) is mainly insoluble in vivo (27), it is possible that the presence of the Hsp33(M172S) in protein aggregates represents a bona fide chaperone-substrate interaction.…”

“…Specifically, we chose the following hslO mutations: Hsp33(Y12E), which is constitutively active in vitro but mainly insoluble in vivo; Hsp33(M172S), activated by high temperature in the absence of oxidizing conditions (27); Hsp33(Q151E), constitutively monomeric and redox-regulated with preference for slowly unfolding protein substrates (38); and Hsp33(1-235), constitutively active as a dimer in vitro (39). The corresponding mutant genes were cloned in a multicopy plasmid under control of an IPTG-inducible promoter and tested for their ability to suppress the growth defects exhibited by the MC4100 ⌬tig ⌬dnaKdnaJ mutant strain.…”

Bile salts act as effective protein-unfolding agents and instigators of disulfide stress in vivo

“…We therefore grew MC4100 wild-type and hslO-deletion strains to mid-log phase, treated the cultures with either CHO or DOC, and took samples at defined time points before and after the treatment. Protein aggregates were separated from the soluble proteins using an established protocol (17). As shown in Fig.…”