Core chaperone families
From Chaperome
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Classification of molecular chaperones
Most molecular chaperones belong to five sequence-related families: Hsp60, Hsp70, Hsp90, Hsp100 and the small HSP (sHsp). They were initially classified according to their sequences and apparent molecular masses. Members of the same family may be located in different cellular compartments. They may vary in time of expression (developmental, stress, etc.) and substrate specificity (nascent proteins, stress-damaged proteins etc).
Hsp60
Definition
The Hsp60 family, also called the chaperonins, comprises two distinct classes, differentiated by sequence-alignment and the need of a lid-like co-chaperone. One class of chaperonins is expressed in bacteria and evolutionarily derived organelles and consists of bacterial GroEL, mitochondrial Hsp60 and chloroplast cpn60. The second class of chaperonins is expressed in the cytoplasm of eukaryotes and in archaea and consists of CCT and the archaeal thermosome (Kusmierczyk and Martin, 2001). The most studied representative of the chaperonin family, is GroEL from E. coli (for a review see, Ellis, 2001; Grallert and Buchner, 2001; Sigler et al., 1998; Thirumalai and Lorimer, 2001; Walter, 2002).
Structure
GroEL is a 14-mer of 57.3-kDa subunits, arranged in two heptameric rings stacked back to back (Braig et al., 1994). The GroEL monomer has three major domains: (1) The apical domain, which binds the substrate and GroES. (2) The intermediate domain, which connects between the apical and equatorial domains and mediates ATP-induced conformational shifts. (3) The equatorial domain, which forms the surface of interaction between the two GroEL7 rings and contains the ATP binding pocket (Braig et al., 1994; Fenton et al., 1994; Xu et al., 1997). The co-factor of GroEL, GroES, is a heptameric ring of 10-kDa subunits, forming a dome-shaped lid that can associate on each side of the GroEL14 (Hunt et al., 1996; Mande et al., 1996). Under the GroES7 cap each GroEL7 ring forms a cavity of ~175,000 Å3 that can accommodate a protein of up to 60 kDa (Lorimer, 1996; Sigler et al., 1998), in a closed hydrophilic environment (Xu et al., 1997).
The folding cycle of GroEL
GroEL passively prevents off-pathway aggregations by interacting with unstable folding or unfolding intermediates and promoting the correct refolding of many different proteins in an ATP-dependent manner (Goloubinoff et al., 1989b; Lorimer, 1996). Binding to GroEL confers stability to the substrate and prevents further aggregations (Buchner et al., 1991; Goloubinoff et al., 1989a). Upon binding of seven ATP molecules, one of the GroEL7 rings undergoes a major conformational rearrangement in which the cavity increases in size from ~85,000 Å3 to ~175,000 Å3 (Roseman et al., 1996; Xu et al., 1997). This shift results in a rotational movement of the apical domains, changing of the hydrophobic surface of the cavity rim into a hydrophilic surface (Xu et al., 1997), which stretches and unfolds of the bound substrate (Shtilerman et al., 1999; Walter et al., 1996; Zahn et al., 1996a). The concomitant binding of the GroES7 lid near the protein-binding sites (Ashcroft et al., 2002) further induces the release of the substrate protein into the central cavity. This allows the protein to complete its correct folding in a confined sequestered cage devoid of illicit interactions with other misfolded substrates (Weissman et al., 1995). Hydrolysis of ATP in the first ring in thought to decrease the negative cooperativity between the two GroEL7 rings, and induce ATP binding in the opposite (trans) ring (Yifrach and Horovitz, 1995), which in turn would induce a conformational shift in the cis ring releasing ADP and GroES. However, the importance of negative cooperativity between the two GroEL7 rings for the chaperoning mechanisms, is questioned by the fact that active a single ring GroEL7 mutants (SR44) can assist the refolding of heat-denatured proteins, as efficiently as a double ring GroEL14 mutants (GroEL44)(Chatellier et al., 2000b).
When the concentration of GroES is limiting, GroES and ADP can be released from the GroEL7 cis-ring before a second GroES7 lid is allowed to bind GroEL14 (Rye et al., 1999). This transition from GroEL14GroES7 (bullet conformation) to GroEL14 (brick conformation) induces substrate refolding (Engel et al., 1995), albeit at a low efficiency (Azem et al., 1995; Ben-Zvi et al., 1998). When, in contrast, the concentration of GroEL and GroES protomers are equal as in the cell (Neidhardt, 1996), two GroES7 can concomitantly bind a single GroEL14 core, an preferentially form a GroEL14(GroES7)2 complexes (football conformation). Under such optimal conditions the chaperonin cycles between the bullet and football conformations, with optimal refolding efficiency (Azem et al., 1995; Ben-Zvi et al., 1998; Grallert and Buchner, 2001).
The substrates and substrate-binding sites of GroEL
As expected from the size of the cavity and from the ability of low-molecular mass proteins to fold spontaneously, the range of molecular masses for typical GroEL-substrates is 20-60 kDa (Houry et al., 1999). Most GroEL substrates expose hydrophobic surfaces and have a net positive charge (Wang et al., 2000) that fit the hydrophilic nature of the substrate-binding ring of GroEL (Xu et al., 1997). Further studies on substrate recognition using intact GroEL or recombinant apical domains named mini-chaperones, revealed that protein binding requires multiple binding sites (Chatellier et al., 2000a; Farr et al., 2000; Wang et al., 2000; Weber et al., 1998). Thus, when GroEL undergoes a structural shift, transitional movements between several binding-sites arranged in a ring, can apply an unfolding force on the substrate protein (Shtilerman et al., 1999; Zahn et al., 1996b). This suggests that GroEL-assisted protein refolding is not direct. It would rather result from the active unfolding of the bound misfolded substrate, which, upon chaperone release, may spontaneously refold according to the dictates of its primary sequence (Anfinsen, 1973).
Hsp70
Definition
Hsp70s (heat shock proteins of the 70kDa size) are a family of molecular chaperones that are highly conserved, exhibiting a high level of sequence identity (45%) across species. With the exception of some archaea, all organisms express several different forms of Hsp70s varying in cellular location and time of expression (Frydman, 2001; Hartl and Hayer-Hartl, 2002). These ubiquitous proteins function in a variety of basic cellular processes such as protein folding and protein translocation across membranes.
The structure and function of Hsp70
Although Hsp70s participate in diverse cellular processes, they share a highly conserved structure. Hsp70 is composed of two major domains, a N-terminal ATPase domain (~40 kDa) and a C-terminal domain containing a substrate-binding cleft covered by a removable lid (~25 kDa). As a molecular chaperone, Hsp70s function to prevent aggregation and ensure the proper folding of proteins through cycles of substrate binding and release regulated by ATP binding and hydrolysis. The ATP-bound form of an Hsp70 has a fast exchange rate for the substrate protein thus having a low affinity for the substrate protein. On the other hand, the ADP-bound form of an Hsp70 binds and releases the substrate protein more slowly, thus having a high affinity for the substrate protein. When ATP is hydrolyzed, a structural rearrangement is induced in the substrate-binding domain of Hsp70 (Flaherty et al., 1990; Flaherty et al., 1994), resulting in the locking of the lid on the protein-binding site (Zhu et al., 1996). Unlocking is triggered by the dissociation of ADP from the chaperone and rebinding of ATP. In the ATP bound state, the substrate-binding site is opened (lid open) and the chaperone can bind and unbind extended hydrophobic segments in unfolded of misfolded proteins (Ha et al., 1999; Mayer et al., 2000; Schmid et al., 1994; Zhu et al., 1996). Hsp40 binding to Hsp70 and the substrate protein stimulates ATP-hydrolysis and the locking of the Hsp70 in an ADP-bound state on the protein segment (Han and Christen, 2003; Laufen et al., 1999; Mayer et al., 2000; Schmid et al., 1994; Zhu et al., 1996). Nucleotide exchange factor binding to substrate-locked ADP-Hsp70, allows ADP and Pi release, which opens the peptide-binding pocket and unlocks the lid (Harrison et al., 1997; Sondermann et al., 2001). The substrate protein is then free to fold into its proper conformation while a new cycle can be triggered by ATP-binding to Hsp70 (Harrison et al., 1997; Packschies et al., 1997).
Substrate specificity of Hsp70s
Hsp70 interacts with many different substrates, such as nascent polypeptides, translocating proteins between cellular compartments and stress-misfolded protein aggregates (Deuerling et al., 1999; Diamant et al., 2000; Zhang and Glaser, 2002). Early studies revealed that Hsp70 binds short peptides in a sequence-dependent manner, and this suggested that Hsp70 recognizes its various substrates by interacting with exposed peptide segments (Flynn et al., 1989). Indeed, the crystal structure of the substrate-binding domain of Hsp70 shows a substrate-binding channel, which can accommodate short peptides (8 amino-acids) in an extended conformation (Zhu et al., 1996). Peptide library scanning revealed that Hsp70 recognition sites are composed of a hydrophobic core with a preference to leucines, and are flanked by positively charged amino acids (Rudiger et al., 1997a; Rudiger et al., 1997b). An algorithm predicts that on average unfolded proteins display a strong Hsp70-binding site every 37 amino acids (Rudiger et al., 1997b). Proteins depending on Hsp70 for their translocation were found to have Hsp70 binding sites in their transit or precursor peptides (Zhang and Glaser, 2002). Despite progress in the understanding of the Hsp70 mechanism, it is unclear how alternating Hsp40-mediated binding and locking of Hsp70, and Nucleotide exchange factor-mediated unlocking and release of Hsp70, to and from a misfolded substrate, can lead to protein translocation or disaggregation and correct substrate folding.
Models of Hsp70 action in Protein Translocation
The mechanism of Hsp70 action in the movement of preproteins through the translcoation channel is a subject of great debate. If mitochondrial Hsp70 activity is blocked by depleting matrix ATP, preproteins are able to slide back and forth in the import channel (Schwarz et al., 1993). The mechanism by which Hsp70 provides unidirectional movement of the preprotein into the matrix is unclear. Two models, which are not mutually exclusive, have been proposed to describe the molecular mechanism of Hsp70 action in the import of preproteins into the matrix of mitochondria: the Brownian motion (trapping) model and the Motor (pulling) model (Neupert, 1997).
The Brownian motion model asserts that translocation of preproteins into the matrix is a spontaneous event (Gaume et al., 1998; Schneider et al., 1994; Ungermann et al., 1994). Domains on the cytosolic face of the mitochondrial outer membrane spontaneously unfold or "breathe" allowing segments of the protein to enter the import channel. The preprotein in transit is free to move back and forth in the channel based on Brownian motion. Once the preprotein emerges from the import channel into the matrix, Hsp70 binds the preprotein and prevents its backslipping. The DnaJ domain containing protein Tim44 is thought to recruit Hsp70 to the import where Hsp70 can interact with the emerging preprotein. Further sponataneous unfolding of folded domains on the cytosolic face of the mitochondrial outer membrane allows additional segments of the preprotein to enter the translocation channel, exposing a new binding stite for another molecule of Hsp70. Thus, the binding of Hsp70 traps the preprotein and prevents its backslipping thereby providing a molecular mechanism for unidirectional movement of the preprotein into the matrix. According to the Brownian Motion model, the translocation of preproteins is efficient if Hsp70 is available at the import channel to bind the preprotein as it emerges into the matrix. According to this model, the role of the DnaJ domain containing protein Tim44 is to recruit Hsp70 to the import channel to provide a high local concentration of Hsp70 for efficient import.
The Motor (pulling) model describes a more active role for Hsp70 in the process of translocation. After the preprotein emerges from the import channel, Tim44-bound Hsp70 binds the preprotein and undergoes a conformational change upon hydrolysis of ATP. Using Tim44 as a membrane anchor, the conformational change of Hsp70 generates a force that will labilize folded domains of the preprotein on the cytosolic side of the outer mitochondrial membrane (Horst et al., 1996, Kronidou et al. 1994, Matouschek et al. , 1997). Thus, Hsp70's pulling force on the protein in transit drives the preprotein into the matrix (Glick, 1995). The interaction of Tim44 and Ssc1 is required for the power stroke of the motor model.
See also Mechanisms
Hsp90
Hsp100
Definition
The Hsp100 family belongs to a larger family of “ATPase Associated with diverse Activities” (AAA+) that contain a conserved ATPase module with a consensus Nucleotide Binding Domain (NBD)(Beyer, 1997). AAA+ participate in diverse cellular processes involving modulation of protein structures or complexes (Lupas and Martin, 2002; Ogura and Wilkinson, 2001). Hsp100 belong to an AAA+ sub-family with one or two NBD domains (Schirmer et al., 1996). While many Hsp100s associate with a protease and thus control protein degradation (Ogura and Wilkinson, 2001; Schirmer et al., 1996), ClpB/Hsp104 does not associate with a protease but is instead implicated with Hsp70 in the solubilization and reactivation of aggregates proteins (Glover and Lindquist, 1998; Goloubinoff et al., 1999; Ben-Zvi and Goloubinoff, 2001).
Structure
Each Hsp100 subunit is composed of three major domains, an N-terminal domain, followed by two Nucleotide Binding Domains, NBD1 and NBD2. The N-terminal domain has a low sequence similarity with other members of the AAA+ family, but has a similar fold (Guo et al., 2002; Li and Sha, 2003; Rouiller et al., 2002). This fold provides a common platform for the recognition of substrate proteins, as well as of adaptor proteins that modify substrate specificity (Dougan et al., 2002). The NBD domains have a conserved ATP-binding and hydrolysis AAA+ module, containing both Walker A (Gx4GKT) and Walker B (Rx6-8h4D) ATP-binding motives (Schirmer et al., 1996). Like other AAA+ proteins, Hsp100 monomers are arranged in a functional hexameric ring (Ogura and Wilkinson, 2001; Schirmer et al., 1996). Various mutations in the conserved ATP-binding sites impair, to various degrees, the ability of Hsp100 to form functional hexamers, while stabilizing the hexamer restores chaperone activity. Hexameric rings are therefore the active oligomeric form of Hsp100 proteins (Diamant et al., 2003; Schirmer et al., 2001). Proteins containing either one or two NBD domains form hexameric ring oligomers and exhibit overall similar types of chaperone activity (Hoskins et al., 2002). Thus, one NBD domain suffices to carry out the basic chaperone function. However, differences in ATP hydrolysis and an important cross-talk between the two NBD domains have been observed in Hsp104 and ClpB (Hattendorf and Lindquist, 2002; Diamant et al., 2003). This indicates that each NBD modulates the ATPase activity of the other, possibly enabling fine-tuning of the chaperone activity. The mechanism that couples ATP-hydrolysis to the chaperone activity in the various Hsp100s still remains unclear. A structural study by electron microscopy of the AAA+ protein p97 showed wide structural differences upon binding of different nucleotides, in particular in the N-terminal and NBD1 domains. This suggests that Hsp100 rings can undergo extensive diaphragm-like movement upon ATP binding and hydrolysis that may induce structural changes in the bound substrate protein (Zhang et al., 2002).
The chaperone activity of Hsp100
In yeast, mutation of the Hsp104 gene has no effect on growth rates at normal or moderately elevated temperatures. But, a functional Hsp104 is obligatory for yeast survival under extreme stresses (Parsell et al., 1991; Sanchez and Lindquist, 1990; Sanchez et al., 1992) and for the resolubilization of heat-shock protein granules following heat-stress (Parsell et al., 1994). Initial genetic evidence suggested that Hsp104 collaborates with Hsp70 in the acquisition of thermotolerance and in the active solubilization of protein aggregates (Sanchez et al., 1993; Vogel et al., 1995). Hsp104 was also implicated in the propagation, as well as in the partial curing, of Sup35 prions (Chernoff et al., 1995; Patino et al., 1996) and this suggested an active role for Hsp104 in the solubilization of prionic aggregates. A combination between Hsp104 and Hsp70 was shown to solubilize and reactivate protein aggregates in vitro (Glover and Lindquist, 1998; Goloubinoff et al., 1999; Shorter and Lindquist, 2004).
Small HSP
Definition
Small Heat Shock (sHsp) family is composed of many different low molecular weigh proteins (15-40 kDa) that shear some sequence homology, mainly in the C-terminal α-crystallin domain and hydrophobic N-terminal region. They are not essential for survival under physiological and stressful conditions but can increases the organism thermotolerance (Thomas and Baneyx 1998).
The structure and function of sHsp
sHsps work in an energy independent fashion, holding onto proteins to prevent irreversible aggregation during stress (as reviewed in MacRae TH, 2005). Under normal physiological conditions sHsp assemble into higher oligomeric complexes, forming a hollow globular structure (the number of subunits composing the oligomer vary in the sHsp family). Buried inside the assembled sHsp are hydrophobic parches, which are assumed to be the substrate binding sites. During stress the sHsp disassemble into dimers, which expose the hydrophobic sites and can bind aggregating proteins. These dimer-substrate complexes may assemble into larger complexes during the stress. How substrates are release from the sHsp is not known one suggested mechanism is the transfer of substrates to other chaperones systems such as DnaK and GroEL (Veirling 1997; Vinger et al 1998; Mogk et al., 2003). sHsp ability to prevent protein aggregation in vitro together with their association with aggregates or inclusion bodies in vivo suggested a role for sHsp in aggregates maintenances in vivo. Indeed, increased expression of sHsp was observed in several protein misfolding diseases.
