Abstract
The molecular chaperone Hsp33 in Escherichia coli responds to oxidative stress conditions with the rapid activation of its chaperone function. On its activation pathway, Hsp33 progresses through three major conformations, starting as a reduced, zinc-bound inactive monomer, proceeding through an oxidized zinc-free monomer, and ending as a fully active oxidized dimer. While it is known that Hsp33 senses oxidative stress through its C-terminal four-cysteine zinc center, the nature of the conformational changes in Hsp33 that must take place to accommodate this activation process is largely unknown. To investigate these conformational rearrangements, we constructed constitutively monomeric Hsp33 variants as well as fragments consisting of the redox regulatory C-terminal domain of Hsp33. These proteins were studied by a combination of biochemical and NMR spectroscopic techniques. We found that in the reduced, monomeric conformation, zinc binding stabilizes the C terminus of Hsp33 in a highly compact, alpha-helical structure. This appears to conceal both the substrate-binding site as well as the dimerization interface. Zinc release without formation of the two native disulfide bonds causes the partial unfolding of the C terminus of Hsp33. This is sufficient to unmask the substrate-binding site, but not the dimerization interface, rendering reduced zinc-free Hsp33 partially active yet monomeric. Critical for the dimerization is disulfide bond formation, which causes the further unfolding of the C terminus of Hsp3 and allows the association of two oxidized Hsp33 monomers. This then leads to the formation of active Hsp33 dimers, which are capable of protecting cells against the severe consequences of oxidative heat stress.
Highlights
Biochemical studies showed that Hsp[33] senses reactive oxygen species through its C-terminal four-cysteine zinc center, with zinc playing an important role for the formation of the correct disulfide bonds and the rapid activation of the chaperone function of Hsp[33] (3)
To create a stably reduced, metal-free Hsp[33] variant, we decided to use a mutant of Hsp[33], in which all cysteine residues had been replaced (Cys-free Hsp33) (5)
We found that the presence of the Hsp[33] gene hslO is required for the high temperature growth of E. coli cells that are constitutively oxidatively stressed due to the absence of thioredoxin reductase (⌬trxB) (Fig. 3). ⌬trxB cells have previously been shown to have a higher resistance against oxidative stress treatment (13), which appears to be at least in part due to the accumulation of oxidized and activated Hsp[33] (2)
Summary
NMR and CD studies of the isolated C-terminal domain and the full-length Hsp[33] wild type protein suggested that only the C-terminal redox switch domain unfolds, while the structure of the N-terminal chaperone domain appears largely unaltered by the oxidation and dimerization process. This mutant protein should allow us to analyze the role of zinc coordination on the activity and conformation of monomeric Hsp[33] without the potential interference of disulfide bond formation.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
