Activation of the Redox-regulated Chaperone Hsp33 by Domain Unfolding

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, ␣-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.

The redox-switch domain of Hsp33 functions as dual stress sensor

“…Hsp33 ox30°C , in contrast, retains much of the α-helical content (30%) found in reduced Hsp33. The CD spectrum of Hsp33 ox30°C was very similar to that of the cysteine-free variant of Hsp33 (Hsp33 Cys− ), which is zinc-free, contains an unfolded zinc-binding domain and is inactive at 30 °C 12 ( Fig. 4a, compare traces 2 and 3).…”

“…We have previously reported that oxidation of Hsp33 at its highly conserved cysteine residues leads to the release of zinc, which causes the unfolding of the zinc-binding domain 12 . To determine whether the lack of Hsp33 activation at 30 °C is due to a lack of zinc release, we used two different assays to monitor the zinc-binding status: a spectrophotometric PAR/PCMB assay, which depends on direct competition for zinc binding between Hsp33's cysteines (Hsp33 red , K a = 2.5 × 10 17 M −1 ) and the chelator PAR (K a = 2 × 10 12 M −1 ) 14 and a fluorescencebased assay, which directly monitors zinc binding in Hsp33 (Fig.…”

“…This unfolding causes either the formation or exposure of hydrophobic surfaces that are probably the substrate-binding site(s) in Hsp33 (Fig. 4a, inset) 12,18 . Hsp33 ox30°C , in contrast, retains much of the α-helical content (30%) found in reduced Hsp33.…”

“…Upon exposure of Hsp33 to oxidative stress, the nearby cysteines form intramolecular disulfide bonds, inducing zinc release 7,11 . Large conformational rearrangements and partial unfolding then lead to the dimerization of Hsp33, a crucial step that fully activates Hsp33's chaperone function 7,12 . The overall activation process of Hsp33 is unusually temperature dependent 7 , and in vivo studies have shown that Hsp33 protects E. coli cells against H 2 O 2 treatment only at elevated temperatures 13 .…”

Reactive oxygen species and cellular oxygen sensing

“…In this assay, cyan and yellow-fluorescent proteins are bridged by part of a bacterial heat shock protein [29,60] which contains redox-sensitive cysteine thiols [61,62]. The thiol groups are reduced at normal conditions but upon oxidation, a disulfide bond is formed that changes the conformation of the hinge domain and separates the YFP and CFP.…”