Characterization of the activation mechanism of the redox-regulated molecular chaperone Hsp33

The redox-regulated molecular chaperone Hsp33 senses oxidative stress through oxidation of the four invariant cysteines of its zinc center. The zinc center occupies the carboxyl-terminal domain of Hsp33. Reactive oxygen species induce the formation of disulfide bonds connecting the nearby cysteines in CXC and CXXC motifs. It has been previously shown that the activation of Hsp33 by hydrogen peroxide is highly temperature-dependent. I report here that elevated temperatures cause the unfolding of the C-terminal regulatory domain. This domain unfolding exposes the substrate-binding site and enables Hsp33 to dimerize into its highly active form. Hsp33 is one of only a few proteins that have been shown to require partial unfolding to gain activity. Oxidative stress-induced disulfide bond formation locks Hsp33 in this partially unfolded, active state. This maintains Hsp33 in its active conformation until reducing, non-stress conditions are restored. An alternative activator of Hsp33 is the antimicrobial oxidant hypochlorous acid. Hypochlorous acid is produced by neutrophils as part of the immune response and by epithelial tissue lining the bronchi and the gut to deter colonization by microbes. In contrast to peroxide, hypochlorous acid causes the unfolding of numerous proteins. I found that while most proteins are inactivated by hypochlorous acid, Hsp33 becomes activated. This hypochlorous acid-induced unfolding of Hsp33 alleviates the need for elevated temperatures for activation by peroxide. It therefore appears that Hsp33 uses the unfolding activity of hypochlorous acid to stimulate its own chaperone activity. Hsp33 is activated by the very conditions that lead to protein unfolding and aggregation inside the cell, ensuring that Hsp33 is activated only when needed. This effectively protects bacteria against these oxidative stress conditions. In fact, other chaperones have been shown to lose their activity under these oxidizing, unfolding conditions. Thus, it is no surprise that nearly all bacteria possess the powerful chaperone Hsp33.