and one Hsp40 member Zuotin (Zuo). Zuo and Ssz form a complex termed ribosome-associated complex (RAC), which is the subject of this study. Recently a functional connection between both chaperone systems NAC and RAC could be shown (Koplin et al., 2010), suggesting a parallel or overlapping pathway in de novo protein folding.
1.5. The Hsp70/40 Chaperone System
1.5.1. The Hsp70 family
Ssz as well as Ssb belong to the class of the 70 kDa heat shock protein (Hsp70) chaperones. Hsp70s are present in most compartments of eukaryotic cells, in eubacteria and in many archaea. They are the central part of a ubiquitous chaperone system and play an important role in different cellular processes (Mayer et al., 2001; Mayer and Bukau, 2005) such as the folding of newly synthesized or misfolded proteins as well as the prevention or reversion of protein aggregation (Deuerling et al., 1999; Goloubinoff et al., 1999; Mogk et al., 1999; Teter et al., 1999). Most quality control functions of Hsp70s require co-chaperones or chaperones of other families and play an important role in stress situations like heat-shock or harsh environmental conditions. All these functions of Hps70 chaperones are driven by their basic property to interact with short hydrophobic peptide sequences in an ATP dependent fashion. Under normal conditions Hsp70s take over housekeeping functions such as disassembly of protein complexes e.g. clathrin coats (Chappell et al., 1987; Ungewickell, 1985), import of proteins into the lumen of the endoplasmic reticulum (ER) (Lyman and Schekman, 1997; Matlack et al., 1999) and the mitochondrial matrix (Voisine et al., 1999), regulation of the heat-shock response and the control of regulatory proteins in cooperation with the Hsp90 system. In E.coli the main Hsp70 chaperone is DnaK which is constitutively expressed at a high level even under normal conditions. Under heat-shock conditions the expression is even higher, upregulated by the heat-shock transcription factor σ32. Higher eukaryotes contain two Hsp70 isoforms, the constitutively expressed Hsc70s and the stress induced Hsp70s which are under the control of the heat-shock response.
Most organisms contain multiple members of the Hsp70 family, some of which co-exist in the same cellular compartment. The yeast Saccharomyces cerevisiae contains four canonical Hsp70s and three fungal-specific ribosome-associated and specialized
Hsp70s. Several studies demonstrated functional specificity among the Hsp70 isoforms, indicating significance of the requirement for multiple Hsp70s (Kabani and Martineau, 2008).
1.5.1.1. Structure of Hsp70 Chaperones
Hsp70 chaperones are structurally highly conserved and consist of two functional do-mains: a ~44 kDa N-terminal nucleotide binding domain (NBD) and a substrate bind-ing domain (SBD) at the C-terminus of about ~25 kDa; the two domains are connected by a short hydrophobic linker (Bukau and Horwich, 1998) (Fig 1.1a). In the case of DnaK, an almost full length structure (aa 1 - 605) has been gained recently by the combination of NMR-RDC & X-ray analysis (Bertelsen et al., 2009) (Fig 1.1b).
Figure 1.1.: Domain Architecture and Structure of Hsp70 -(a)Domain organization of DnaK with nucleotide binding domain (NBD) and substrate binding domain (SBD) (b) Secondary structure representation of E.coli DnaK (PDB code 2KHO). In gray, secondary structure representation of SBD (393-605) and in blue, secondary structure representation of NBD (1-366) with lobes I and II, formed by subdomains A and B, respectively.
The nucleotide binding domain of Hps70s has a similar fold as monomeric actin,
con-1.5. The Hsp70/40 Chaperone System sisting of two structural lobes forming a V-like shaped domain with a deep
nucleotide-binding cleft. Each lobe can be further divided into two subdomains IA, IB and IIA, IIB (Fig 1.1b). The binding pocket for the nucleotide and the required Mg2+ and K+ ions is formed at the bottom of the cleft by all four subdomains and the cross-connecting helices, contacting the adenosine nucleotide with two α- and β-binding loops (Flaherty et al., 1990). By X-ray structure analysis of the ATPase domains of bovine, human and bacterial Hsp70 only subtle conformational changes were found in response to nucleotide binding or hydrolysis (Flaherty et al., 1994; Harrison et al., 1997; Sondermann et al., 2001). In contrast, NMR data revealed a high flexibility in the NBD with an opening and closing motion of the nucleotide binding cleft (Zhang and Zuiderweg, 2004). Amide-Hydrogen-Exchange experiments on full length DnaK supports the findings by NMR analysis, revealing a significant flexibility in all four subdomains of the NBD relative to each other (Rist et al., 2006). The peptide bind-ing domain of DnaK is divided into a β-sandwich subdomain which consists of two four-stranded α-sheets with four loops protruding upwards (two inner and two outer loops) and an α-helical extension that functions as a lid for substrate enclosure (Zhu et al., 1996). Binding of peptides occurs in between the loops L1,2 and L3,4 by forming a direct hydrogen bond with the peptide substrate backbone (Bertelsen et al., 1999;
Morshauser et al., 1999; Pellecchia et al., 2000; Stevens et al., 2003). Helix B consti-tutes a lid which closes the cavity through a salt bridge and two hydrogen bonds to the outer loops L3,4 and L5,6. The function of the subdomain formed by the distal part of Helix B and the 3 additional helices C, D, and E is still unknown.
1.5.1.2. Hsp70 functional cycle
To assist folding Hsp70 chaperones undergo repeated cycles of substrate binding and release between two nucleotide states (Bukau and Horwich, 1998; Hartl and Hayer-Hartl, 2002). During these cycles Hsp70 alternates between two different structural states, depending on the phosphorylation state of the nucleotide bound (Fig 1.2). In the ATP bound state, the lid over the peptide-binding cleft is in an open conformation:
The affinity for substrates is low, but the association and dissociation rates of the substrate are high (low-affinity state). In contrast, in the ADP bound form, the binding pocket is closed, resembling a high-affinity state for substrates, but has low association and dissociation rates.
Figure 1.2.: Functional ATPase cycle of Hsp70 The cycle involves the high-substrate-affinity ADP state and the low-high-substrate-affinity ATP state. ATP hydrolysis is catalyzed by the syner-gistic action of J-domain-proteins of the Hsp40 family and the substrate. Nucleotide-exchange factors (NEF) accelerate nucleotide release and the subsequent ATP binding leads to substrate release.
However, the basal or substrate stimulated ATP hydrolysis rate is too low to be of physiological relevance (Flynn et al., 1989; Jordan and McMacken, 1995; McCarty et al., 1995). Therefore, J-domain proteins and nucleotide exchange factors (NEFs) regulate the cycling of Hsp70. Upon substrate binding to Hsp70 in the ATP-bound state, interaction with JDP stimulates ATP hydrolyzes of Hsp70, facilitating a tight peptide capture (Mayer et al., 2000). Substrate release from Hsp70 requires exchange of the bound ADP to ATP which is catalyzed by NEFs (Harrison et al., 1997). Rebinding of ATP resets Hsp70 to its initial low substrate affinity status, completing the reaction cycle.
1.5.2. Hsp40 co-chaperone family
The J-domain (Hsp40) protein family, is much larger and more heterogeneous than the Hsp70 family (Laufen et al., 1999; Sahi and Craig, 2007). Zuotin the second component of the ribosome-associated complex (RAC) belongs to this family. All Hsp40s use their
1.5. The Hsp70/40 Chaperone System J-domain to stimulate the ATPase activity of their Hsp70 partners. The J-domains
consist of about 65 amino acids, forming an elongated domain composed of helical bundles with an absolutely conserved Hsp70 interacting motif His-Pro-Asp (HPD) exposed on a loop at one end (Qian et al., 1996; Cheetham and Caplan, 1998; Hennessy et al., 2005; Qiu et al., 2006).
Hsp40 proteins first act as a chaperone by binding unfolded peptides and forwarding them to their Hsp70 partner. Concurrently they stimulate the ATP activity of the Hsp70s, resulting in a more than 100-fold stimulated ATP hydrolysis compared to basal rate (Laufen et al., 1999). Despite the highly conserved J-domain, Hsp40s differ in their domain organization and localization within the cell (Walsh et al., 2004). Depending on their domain structure Hsp40s are organized in 3 different types (Fig 1.3).
Figure 1.3.: Structural classification of J-proteins Representation of type I, II and III J proteins from yeast aligned according to the N-terminus of the mature protein. The gray boxes represent each polypeptide and show the scale of the J domain (J, pink), glycine-rich region (G, yellow) and zinc-finger domain (Zn-finger, green). CTD, carboxy-terminal domain
Type I Hsp40 proteins have a compact helical J-domain that is linked by a glycine-rich region to a zinc-finger domain followed by a C-terminal domain. Type II proteins have a J domain linked by a glycine-rich region to a C-terminal domain, whereas type III proteins have the J-domain only. Type I and type II proteins are functionally similar. Both bind to non-native substrates for presentation to their Hsp70 partners and are found in the cytosol. In contrast, type III proteins are found at diverse intracellular sites such as the ER, mitochondria or attached to ribosomes. None of them has been shown to bind non-native polypeptides. Therefore, functionality as chaperone is unlikely. Zuotin fromS. cerevisiaeis a ribosome bound Hsp40, belonging to the type III J-domain proteins.