Classes of molecular chaperones

General classifications: Molecular chaperones are central to the cellular proteostasis network. They facilitate protein biogenesis by assisting folding, translocation and assembly of newly made proteins or guide the resolubilization of aggregated proteins and help to deposit or degrade terminally misfolded polypeptides (Fig. 9A) (GERSHENON &GIERASCH, 2011; HARTL et al., 2011; MILLER et al., 2015). Chaperones are defined as proteins that interact with, stabilize or help other proteins to acquire their functional conformation. These factors neither provide steric information for folding nor are they present in the final structure of a protein (HARTL, 1996; HARTL &HAYER-HARTL, 2009). Several classes of structurally unrelated chaperones exist and members of these families are known as stress proteins or heat shock proteins (HSPs), as they are upregulated under stress conditions such as heat (RICHTER et al., 2010). Although most chaperones form higher oligomers, they are classified according to their monomeric molecular weight into the conserved families of Hsp40s, Hsp60s (chaperonins), Hsp70s, Hsp90s, Hsp100s, and small heat shock proteins (sHsps) (Fig. 9) (HARTL et al., 2011).

Chaperones are present in every cellular compartment e.g. the nucleus, ER or mitochondria. Some chaperones are constitutively expressed (e.g. Hsc70 or Hsc90), whereas others are upregulated upon stress (e.g. sHsps). In addition, species-specific chaperones exist like the bacterial Trigger Factor (TF) or the archaean and eukaryotic nascent polypeptide-associated complex (NAC) (KIM et al., 2013) that do not belong to any of the classic families. Based on the interaction with client proteins, chaperones are classified as holdases, foldases and disaggregases. Holdases represent ATP-independent proteins (e.g. sHsps or Hsp40s) which can recognize and stabilize partially folded proteins. They prevent protein aggregation and present client proteins to foldases and represent a first line of defense until foldases or disaggregases are available (HASLBECK & VIERLING, 2015).

Foldases (e.g. Hsp60s, Hsp70s or Hsp90s) are directly involved in protein remodeling in an ATP- and cofactor-dependent manner. Disaggregases (e.g. Hsp100) are also ATP-dependent and solubilize aggregates in combination with their transfer to holdases or foldases (SLEPENKOV & WITT, 2002; BÖSL et al., 2006;

DIAZ-VILLANUEVA et al., 2015).

Global recognition of partially unfolded proteins through exposed hydrophobic patches allows binding of molecular chaperones to a large variety of substrates: folding or assembly intermediates, misfolded or denatured polypeptides, proteins that have been translocated to a cell compartment or partially unfolded substrates of the proteolytic machinery (Fig. 9A) (MAYER, 2010; RICHTER et al., 2010). Binding to hydrophobic regions temporarily blocks protein aggregation, while ATP hydrolysis is important to allow client remodeling. However, the precise contribution of ATP-dependent chaperones to the folding process is still unknown. In the case of Hsp70s it is proposed that the energy from ATP hydrolysis is used to unfold polypeptides that, upon dissociation from the chaperone, can spontaneously refold or reenter the chaperone cycle (Fig. 9C) (SHARMA et al., 2010).

Although Hsp60s and Hsp70s both operate by this basic mechanism, they differ fundamentally in their precise mode of action: Hsp70s release their substrates for folding into the bulk solution upon ATP hydrolysis, whereas the cylindrical chaperonins allow folding of single protein molecules enclosed in a cage (Fig. 9B/C). The two systems act sequentially as Hsp70s interact with nascent polypeptides followed by the action of chaperonins that promote final folding of those proteins that fail to reach their native state by cycling on Hsp70s alone (LANGER

et al., 1992; FRYDMAN et al., 1994; HARTL et al., 2011).

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Figure 9: Classes of molecular chaperones. A) Molecular chaperones interact with unfolded (U) proteins that fold via increasingly structured intermediates (I1, I2) into their native state (N). B) Functional cycle of Hsp60 chaperonins, exemplarily shown by GroEL/ES. Non-native protein binds to GroEL; upon ATP and GroES association the protein is encapsulated. ATP hydrolysis in one ring results in Gro-ES and substrate release from the opposite ring; (D) indicates the ADP state, (T) that of bound ATP. C) Functional cycle of Hsp70 and its co-chaperones Hsp40 and nucleotide exchange factor (NEF). Hsp40 interacts with the substrate and delivers it to the Hsp70. Upon ATP hydrolysis triggered by Hsp40, NEF releases ADP, ATP is bound by the Hsp70, which leads to release of the substrate. D) Functional cycle of Hsp90s and cofactors. Yeast cochaperones Sti1/Hop binds Hsp70 and Hsp90 thus forming a complex that also contains a PPIase and inhibits the ATPase of Hsp90s. The client protein is transferred from Hsp70 to Hsp90, Sti1/Hop is released once ATP and a further co-chaperone (p23) are bound by the Hsp90. ATP hydrolysis leads to client release. E) Function of Hsp100 disaggregase (ClpB/Hsp104). Protein aggregates are dissolved by actively pulling them through a central channel of the hexameric Hsp100. Each protomer contains two ATPases. Refolding can occur upon release, often in cooperation with other chaperones. F) Small Hsps (sHsps) form oligomeric complexes that are activated (red) e.g. upon heat, which leads to their dissociation into smaller oligomers. sHsps bind non-native proteins as a complex and release substrates in cooperation with other ATP-dependent chaperones such as Hsp70 (adapted from RICHTER et al., 2010).

Heat shock protein families: Chaperones are classified according to their molecular weight into the families of Hsp40s, Hsp60s, Hsp70s, Hsp90s, Hsp100s, and sHsps (Fig. 9). Examples of each chaperone family and their mode of function together with cofactors will be described in the following part.

Hsp60s (Fig. 9B) or chaperonins (HEMMINGSEN et al., 1988) are ATP-dependent foldases that can be divided into two subgroups: Group I members are present in bacteria (e.g. bacterial GroEL) and eukaryotic organelles;

they possess seven-fold symmetry and assemble into a barrel-like structure composed of two rings of identical subunits (HAYER-HARTL et al., 2016). ATP binding triggers conformational rearrangements of apical domains of the Hsp60 followed by co-chaperone (Hsp10, GroES) binding, that caps one side of the barrel (XU et al., 1997) to encapsulate the substrate in a nano-cage (CHEN et al., 2013) and to promote protein folding (SIGLER et al., 1998). The current model suggests that GroEL-ES promotes folding through cycles of substrate binding, encapsulation and release (HAYER-HARTL et al., 2016). Group II Hsp60s are found in archaea and the eukaryotic

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cytosol (e.g. CCT/TRiC) and are homo- or hetero-oligomers forming an eight-fold double barrel structure (LOPEZ et al., 2015). Unlike group I, members of group II do not function together with co-chaperones but contain a built-in lid that undergoes an iris-like motion to promote protein folding (BOOTH et al., 2008).

Hsp90s (Fig. 9D) are abundant and conserved ATP-dependent foldases that, together with Hsp70s and further co-chaperones, facilitate late-stage folding and maturation of proteins (LI &BUCHNER, 2013). Hsp90 exist in bacteria (HtpG) as well as in eukaryotes, e.g. the ER resident Grp94 or the mitochondrial TRAP1 (JOHNSON, 2012), but are absent in archaea. As Hsp90 substrates are in most cases substantially folded proteins they were termed “clients” (PEARL &PRODROMOU, 2006). Hsp90s share a conserved domain structure consisting of an N-terminal nucleotide-binding domain, a charged linker, a middle domain that is involved in client binding and a C-terminal dimerization domain via which Hsp90s form homodimers (JENG et al., 2015). In the apo state, Hsp90s represent a V-shaped open dimer with the N-domains separated by >100 Å (SHIAU et al., 2006) while the ATP-bound conformation adopts an N-terminally closed dimer (LAVERY et al., 2014). Clients bind to the open conformation followed by ATP and cofactor interaction and ATP hydrolysis which triggers client and ADP release.

Members of the Hsp100 family (Fig. 9E) belong to the large AAA superfamily of ATPases associated with diverse cellular activities (NEUWALD et al., 1999) and are present in bacteria (Clp), yeast (Hsp104) and plants, but are absent in the cytosol of higher eukaryotes (KIM et al., 2013). Hsp100 forms a hexameric ring that is stabilized by nucleotide binding and functions as an ATPase in cooperation with Hsp70/40 systems (DnaK/J in bacteria and Ssa/Ydj1 in yeast) (GOLOUBINOFF et al., 1999) to recover functional proteins from aggregates.

Hsp70s direct Hsp100s to aggregates (WINKLER et al., 2012) from which Hsp100s extract polypeptides using an ATP-driven power stroke threading the polypeptide through the central channel of the hexamer (WEIBEZAHN

et al., 2004). Thereby, the disaggregated protein gets another attempt to fold or it can be delivered to the cellular degradation machinery (HASLBERGER et al., 2010).

Small heat shock proteins (Fig. 9F) are ubiquitous molecular chaperones that can be found in all domains of life.

They are quite diverse and have evolved independently in metazoans, plants and fungi (HASLBECK &VIERLING, 2015). sHsps associate with a variety of non-native proteins in an ATP-independent manner and prevent irreversible aggregation, thereby representing a first line of defense. The size of sHsp monomers ranges from 12 to 42 kDa and most sHsps form large oligomeric assemblies which are characterized by a conserved beta-sandwich alpha-crystalline domain that is flanked by variable sequences. sHsp oligomers can be activated by a shift towards smaller species, often dimers. These interact with unfolded substrates which are subsequently refolded, e.g. by Hsp70 chaperones (HASLBECK &VIERLING, 2015).

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