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Chaperonin (Hsp60) Proteins: The Molecular Cages

Hsp60 family of chaperones or chaperonins is key components of cellular chaperone machinery. They are the most conserved and ubiquitous class of a molecular chaperone present in the plastids, mitochondria, and cytoplasm of all eukaryotes and eubacteria. Chaperonins form a part of elaborate, co-operative network of chaperones that ensure correct folding of newly synthesized or stress denatured proteins (1, 9, 10). The primary sequence of several polypeptides in the cell consists of multiple domains with α/β fold. Such proteins with complex topologies require assistance of these specialized folding machines. Chaperonins form oligomeric, high molecular weight complexes of ~800 kD. It forms a large cage like structure formed by two heptameric rings, each enclosing a central cavity. The heptamer is made of similar subunits, each having a molecular weight of 57 kD (2).


Classification of Chaperonin: Group I and Group II

The chaperonins are classified into two groups that are structurally similar and quite diverse in sequence (7). Both the groups divide along two distinct evolutionary lines. The group I chaperonins are found in prokaryotes and endosymbiotic organelles such as mitochondria and chloroplasts. Group II chaperonins exist in archaea and eukaryotic cytosol (1). Group I proteins include GroEL in bacterial cytosol, Hsp60 in mitochondria and Rubisco binding protein (RuBisCoBP) in chloroplasts (2). They consist of double-torous shaped complexes, composed of either 1 or 2 (RuBisCo) subunits (4, 7). Group II chaperonins constitute Thermosome/TF55 in archaea and TRiC/CCT in the eukaryotic cytosol. They share the double-torous structure with their group I counterparts but are composed of 2-3 subunit types in archaea and 8 (Cct1-8) subunit types in eukaryotes (6, 7). The members of the two groups function through a similar overall mechanism but differ in the method of substrate encapsulation, which is evident to inspection of their architectures: Group I chaperonins employ a detachable “lid” structure (GroES/Hsp10) that binds in an ATP-dependent fashion, whereas group II chaperonins employ a built-in protrusion structure which may be either marginate or open upward in a polypeptide-accepting state and that then closes when the ring binds ATP to produce the folding-active encapsulated state (3,6, ). Despite the different encapsulation mechanisms, the ATP-directed reaction cycles of the two families of machine appear to be fairly similar, directed by virtually identical equatorial ATP-binding domains (4,7).

Group I chaperonin: Structure and mode of action

Group I Chaperonin: Reaction cyclechaperonin
Domain Organisation of Chaperonins

A good example of group I chaperonins is the paradigmatic Escherichia coli GroEL/GroES system. It consists of 14 subunits arranged in a double heptameric ring complex and requires a ring-shaped co-factor GroES for its function. Each heptamer encloses a central cavity that forms the folding chamber for the polypeptide substrate (3, 8). The constituent subunits of the heptamer contain three domains; the equatorial domain harboring the ATP binding site, a substrate binding apical domain and a middle hinge-domain that enables communication between equatorial and apical domain. The equatorial domain is more or less conserved among paralogous subunits (3, 4, 8). Most of the sequence divergence lies in the apical domain that contains the substrate binding sites. The apical domain forms the opening the GroEL cylinder and exposes a number of hydrophobic residues towards the ring cavity for substrate binding (4).

The ATP binding to GroEL occurs through allosteric mechanisms (5). ATP binds to the subunits within a ring via concerted mechanism in a positively cooperative manner (3). However, negative cooperativity exists for sequential binding of ATP between the rings. This ensures asymmetric behavior of the complex as a two stroke machine (3,4). Prior to ATP binding both the rings are in the tense (T) state. Negative cooperativity allows the nucleotide to bind to cis ring converting it to relaxed (R) state and lowering the nucleotide affinity of the trans ring (5). T state has high affinity for substrate and R state has a lower affinity. Unfolded polypeptide substrates exclusively having either α-helical or β-sheets bind to the exposed hydrophobic patches at the apical domain of GroEL in T state (4). Nucleotide binding results in elongation of the oligomeric structure and twisting of the apical domains resulting in occlusion of the hydrophobic binding sites. Hence ATP binding causes substrate release and binding of GroES (3,4,5). This allows establishment of intramolecular interactions in protected aggregation free environment (3). Hydrolysis of ATP in the cis ring followed by nucleotide binding in the trans ring cause dissociation of GroES and substrate release. Complete folding of the polypeptide to native state prevents further interaction with the hydrophobic sites, otherwise the substrate is primed to next round of chaperone mediated folding (3,4).

Group II chaperonin: Structure, mode of action and physiological significance

Group II Chaperonin: Reaction cycle

In contrast to GroEL which is stress induced (10), TRiC is expressed constitutively and is required for folding of essential proteins (1). TRiC interacts with ~10% of newly synthesized proteins and the list of identified substrate include – cytoskeletal proteins actin and tubulin, cyclin E, Cdc20 and Von Hippel-Lindau tumor suppressor (VHL). Most of the substrates were found to share tryptophan-aspartic acid (WD) repeats, which form a β-propeller domain. Some of TRiC substrate exceeds 100 kD size, and many of them cannot be folded by classical prokaryotic and eukaryotic chaperones. Hence it is quite likely that TRiC and some of its substrates co-evolved concurrently, and TRiC binds and promotes folding of individual domains of these large proteins (3). An interesting feature of many TRiC substrates is that they function as a part of higher-order complexes (2). Release of such a substrates from TRiC only in the presence of their partner proteins and is coupled to an assembly of subunits in complex. This strategy provides a quality control to prevent the release of unassembled component that might have a potentially harmful dominant negative effect on the functioning of the complex (3). Unlike the bacterial chaperonin GroEL, TRiC can bind co-translationally to nascent chains as they emerge from the ribosomes. Substrate binding to TRiC requires the assistance of upstream chaperones. The emerging polypeptide chain is transferred from the ribosome to TRiC via either the chaperone GimC or the Hsp70 chaperone machinery via Prefoldin (3,6). Prefoldin is a jellyfish-shaped component with six tentacles each composed of α-helical coiled-coil. Each tentacle tip contains a hydrophobic substrate binding surface. Although not essential it appears to enhance the efficiency of the overall folding of its preferred substrates, actin and tubulin, presumably by providing efficient delivery (6).

The overall domain architecture TRiC subunits, and its mode of action is similar to GroEL system but unlike the group I chaperonin it does not contain the lid component. Instead, the apical domain of TRiC has a long extension, which forms α-helical protrusion (3). This extension is highly conserved in the TCP1 family and contains hydrophobic residues that act as an substrate-binding sites. The extensions arch over the central channel to form substrate-binding surface and a act as a lid. Like GroEL, TRiC also shows dual cooperativity in nucleotide binding (2). However, ATP binding to the asymmetrically arranged CCT subunits occurs in a sequential rather than a concerted ATP binding mechanism within a ring. This phenomenon may be associated with an ordered sequential release of substrate protein from binding sites on different subunits of the heterooligomeric CCT ring, contrasting with a simultaneous release from all subunits in the GroEL system (6). The mode of TRiC substrate binding and folding is still debatable, and no clear mechanism has been proposed yet. The sequence divergence of TRiC subunits has expanded the range of possible motifs that are accepted in substrates beyond the simple hydrophobic sites offered in group I chaperonins. (2,6) Studies using actin have indicated several possibilities, ranging from polar and charged sequences surface-exposed on the native protein to delineated, hydrophobic sequences. The specificity of TRiC function indicates that TRiC operates by a fundamentally different mechanism of folding (6). Recent experiments have shown substrates bind to TRiC in either a quasi-native or unstructured conformation. Mutations in TRiC are linked to several pathological states such as sensory neuropathy, tumorigenicity and formation of poly glutamine aggregates, highlighting its essential role in the cell (3).

Hsp60 as signaling molecules

The first clue that chaperonins may have roles other than protein folding was provided by the report suggesting that Cpn60.2 of Mycobacterium tuberculosis (Hsp65) had the ability to stimulate human monocytes to secrete the early response proinflammatory cytokines, interleukin -1b and tumor necrosis factor-α. Cpn60 homologues from a range of bacteria were shown to elevate the mRNA levels of several proinflammatory cytokines in macrophage cultures (12). It is now quite evident that many bacterial species ranging from Actinobacillus actinomycetemcomitans, Borrelia burgdorferi, Helicobacter pylori, Haemophilus ducreyi to L pneumophila all express Cpn60 on their outer surfaces, and it has been proposed that Cpn60 acts as an adhesion which enables bacteria to bind to host cells and mediating host cell invasion or inhibit the growth of various other cell populations (11). In humans, extramitochondrial distribution has been reported for hCpn60 where it acts as a receptor for various ligands such as p21 and HIV gp41 (11,13). Further it has been reported that Cpn60 has a high affinity for lipid monolayers and bilayers and can insert into such structures through its hydrophobic C terminus. Cpn60 also acts as a potent stimulator of immune response and may have a role as a danger signal (12,13). Exposure to cpn60 alerts the adaptive immune system and stimulates the release of cytokines and interferones (11,13). However, due to a high degree of sequence conservation between Hsp60 orthologs, such as activation of immune response sometimes result in autoimmune disorders causing bone resorption, infertility and embryo loss. Additionally, through its protein folding function Hsp60 has been shown to influence may prion diseases. Bacterial cpn60 interacts with the normal cellular PrP isoform and converts it to the pathogenic PrPSc isoform that forms the disease causing fibrillar aggregates (11,12).


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Database Statistics

Total Number of Genomes
Numbers in Bacteria
Numbers in Archaea
Numbers in Protists
Numbers in Fungi
Numbers in Algae
Numbers in Plants
Numbers in Animals
Total Number of Hsp60 Proteins