Activation of the redox-regulated molecular chaperone Hsp33 - A two-step mechanism

Background: Hsp33 is a novel redox-regulated molecular chaperone. Hsp33 is present in the reducing environment of the cytosol and is, under normal con ditions, inactive. The four highly conserved cysteines found in Hsp33 const itute a novel zinc binding motif. Upon exposure to oxidative stress, Hsp33' s chaperone activity is turned on. This activation process is initiated by the formation of two intramolecular disulfide bonds. Recently, the 2.2 Angs trom crystal structure of Hsp33 has been solved, revealing that Hsp33 is pr esent as a dimer in the structure (Vijayalakshmi et al., this issue, 367-37 5 [1]). Results: We show here that oxidized, highly active Hsp33 is a dimer in solu tion. In contrast, reduced and inactive Hsp33 is monomeric. The incubation of reduced Hsp33 in H2O2 leads to the simultaneous formation of two intramo lecular disulfide bonds and the concomitant release of zinc. This concentra tion-independent step is followed by a concentration-dependent association reaction. The dimerization of Hsp33 requires highly temperature-sensitive s tructural rearrangements. This allows Hsp33's activation process to be grea tly accelerated at heat shock temperatures. Conclusions: The regulation of Hsp33's chaperone function is highly sophist icated. On a transcriptional level, Hsp33 is under heat shock control. This increases the concentration of Hsp33 under heat and oxidative stress, a pr ocess that favors dimerization, a critical step in Hsp33's activation react ion. On a posttranslational level, Hsp33 is redox regulated. Dimerization o f disulfide-bonded Hsp33 monomers leads to the formation of two extended, p utative substrate binding sites. These sites might explain Hsp33's high and promiscuous affinity for unstructured protein folding intermediates.