The redox-regulated chaperone holdase Hsp33

Hsp33, a 33 kDa heat shock protein, is highly conserved in bacteria and some eukaryotic parasites such as Leishmania. It is constitutively expressed under non-stress conditions (Chuang & Blattner 1993, Jakob et al 1999).

The presence of four absolutely conserved cysteine residues involved in metal binding at the C-terminus of the protein was a unique feature among all known Hsps. Due to the extensive work of the Jakob Lab during the past two decades, this zinc-coordinating cysteine motif was characterized as a universal posttranslational redox switch in proteins, connecting increased ROS levels to functional changes in proteins (Graf et al 2004, Groitl & Jakob 2014, Ilbert et al 2007, Jakob et al 2000). Chaperone-inactive Hsp33 coordinates zinc via four thiol groups, which upon oxidation-ind uced disulfide bond formation and zinc release, turn Hsp33 into a potent chaperone holdase (Graumann et al 2001, Ilbert et al 2007, Jakob et al 2000). Activation of Hsp33 requires a combination of oxidative stress and increased protein unfolding that can be induced by elevated temperatures or bile salts (Cremers et al 2014, Cremers et al 2010, Graumann et al 2001). Bacteria encounter these stress conditions

16 during fever or when passing through the gastrointestinal tract (Cremers et al 2014). Moreover, another fast activator of Hsp33 is HOCl, which simultaneously causes protein unfolding and strongly oxidati ve conditions (Winter et al 2008). HOCl is produced by cells of the innate immune response to kill bacteria (Miller & Britigan 1997) or used as the active component of general disinfectants such as bleach.

Hsp33 consists of two domains, a compactly folded N-terminal domain (blue) and a C-terminal redox-sensing domain (yellow), connected by a flexible linker region (Figure 1.3, green). As shown in Figure 1.3, under reducing non-stress conditions the four absolutely conserved cysteines of

Figure 1. 3: Mechanism of Hsp33’s chaperone activation

In its inactive form E. coli Hsp33 is a reduced and tightly folded monomer (combined from Protein Data Bank ID: 1I7F and 1XJH). The redox-sensing domain within the C-terminus (yellow) consists of four absolutely conserved cysteine thiols coordinating a zinc ion with high affinity. A flexible linker region (green) covers parts of the N -terminal domain (blue) and connects the C-terminal domain to it. Consecutive oxidation of the for thiols groups accompanied by protein unfolding conditions (e.g. heat) leads to the formation of a two disulfide bonds, zinc release, dimerization and unfolding of the C -terminal domain and linker region, presenting the hydrophobic surface of the N--terminal platform. In the chaperone-active dimeric form (modified from Protein Data Bank ID:

1I7F), Hsp33 uses a combination of its partly disordered C-terminal domain and linker region together with the hydrophobic surface of the N-terminal domain to bind unfolded client protein. Hypochlorous acid (HOCl) induces protein unfolding and oxidative conditions simultaneously and is therefore a fast activator of Hsp33’s chaperone -active form.

17 the C-terminal domain arranged in a C²³²-X-C²³⁴-Xⁿ-C²⁶⁵-XY-C²⁶⁸ motif are engaged in the tetrahedral coordination of one Zn²⁺ ion (red sphere), stabilizing the C-terminal domain (Graf et al 2004, Janda et al 2004). The highly charged ~52 amino acid linker region, folded in three α-helices, covers the largely hydrophobic four-stranded β-sheet platform of the N-terminal domain (Figure 1.3) (Janda et al 2004, Vijayalakshmi et al 2001).

Under oxidative stress conditions, the distal cysteines Cys²⁶⁵/Cys²⁶⁸ engage in disulfide bond formation, release the zinc ion and induce unfolding of the C-terminal domain, which further destabilizes the upstream linker region and converts it into a thermolabile folding sensor (Ilbert et al 2007, Vijayalakshmi et al 2001). To activate this dynamic chaperone-inactive oxidation intermediate, a second stressor, namely protein unfolding conditions (e.g.

elevated temperatures or bile salt), is required to shift the equilibrium of the linker region towards the fully unfolded conformation. Now, the proximal cysteines Cys²³² and Cys²³⁴ are available for oxidation, forming a second disulfide bond, accompanied by unfolding of the linker region (Graf et al 2004, Graumann et al 2001, Ilbert et al 2007). Kinetically fast oxidants (e.g.

HOCl), however, rapidly form the second disulfide bond even in the absence of conditions leading to protein unfolding (Winter et al 2008). In the unfolded conformation, two oxidized Hsp33’s dimerize and thus form a highly active chaperone (Figure 1.3).

Unfolding of the linker region exposes a hydrophobic surface in the N-terminal platform. It was assumed that this area serves as a binding site for unfolded client proteins under stress conditions (Ilbert et al 2007). A more recent study now showed that oxidized, chaperone-active Hsp33 uses its own disordered linker region to interact with early protein-unfoldi ng intermediates (Reichmann et al 2012). These results strongly suggest that apart from the N-terminal platform, the flexible linker region is involved in substrate binding. Indeed, a new study using unnatural amino acids incorporated into Hsp33 to enable in vivo crosslinking and fluorine-19 nuclear magnetic resonance (¹⁹F NMR) spectroscopy, revealed a composite client-binding site that consists of polar residues from the flexible linker

18 region as well as nonpolar residues from the N-terminal β-sheet platform surface of Hsp33 (Groitl et al 2016).

Hsp33 uses an intriguing activation mechanism that requires substantial unfolding of its secondary structure to gain chaperone function. Protein

Figure 1. 4: Hsp33’s redox-mediated chaperone function

Oxidative stress can induce substantial structural changes in proteins, causing protein unfolding and aggregation. Bacteria employ the inducible molecular chaperone Hsp33 to sequester unfolding intermediates and prevent accumulation of toxic protein aggregates. Under non-stress conditions, the C-terminal redox-sensing domain (yellow) and flexible linker region (green) of chaperone inactive Hsp33 are well folded, harboring a zinc ion coordinated by four reduced cysteine residues. Upon exposure to oxidative stress, protein unfolding conditions, and depleted ATP levels, Hsp33 undergoes oxidative disulfide bond formation, zinc release and massive structural rearrangements, including significant unfolding of the C-terminal redox-sensing domain and flexible linker region, turning into chaperone active Hsp33. In this partially intrinsically disordered conformation, Hsp33 is able to tightly bind early unfolding protein intermediates and prevent their aggregation. Upon return to non-stress refolding conditions, Hsp33’s thiols become reduced. However, full inactivation of the chaperone functions requires the interaction with the DnaK/DnaJ/GrpE system, which receives the client protein and promotes full inactivation of Hsp33. The KJE system uses the now recovered ATP levels to refold the client proteins to their native structure.

19 unfolding was previously thought to be detrimental for the function of most proteins. However, Hsp33 utilizes unfolding to activate a specific function, making it the founding member of a new category of so-called conditionally disordered chaperones (Bardwell & Jakob 2012, Jakob et al 2014, Reichmann et al 2012).

The oxidative activation of Hsp33’s chaperone function is fully reversible.

Upon return to non-stress reducing and protein refolding conditions, oxidized Hsp33 dimers are reduced by the cellular glutaredoxin and thioredoxin system yet remain bound to their client proteins (Hoffmann et al 2004). Only when ATP levels are restored, ATP-dependent chaperone foldases composed of the bacterial DnaK/DnaJ/GrpE (Hsp70) system mediate the release of Hsp33’s client proteins and its full inactivation (Hoffmann et al 2004, Reichmann et al 2012). Subsequently, the KJE system supports the refolding of client proteins to the native state.

No Hsp33 homologues are known in higher eukaryotes. Recent evidence now suggests that the highly conserved eukaryotic Get3 ATPase moonlights as a redox-regulated chaperone under ATP-depleting stress conditions, such as oxidative stress (Powis et al 2013b, Voth et al 2014).