Natural chaperonin of the hyperthermophilic archaeum, Thermococcus strain KS-1: a hetero-oligomeric chaperonin with variable subunit composition

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Prefoldin is a jellyfish-shaped hexameric co-chaperone of the group II chaperonins. It captures a protein folding intermediate and transfers it to a group II chaperonin for completion of folding. The manner in which prefoldin interacts with its substrates and cooperates with the chaperonin is poorly understood. In this study, we have examined the interaction between a prefoldin and a chaperonin from hyperthermophilic archaea by immunoprecipitation, single molecule observation, and surface plasmon resonance. We demonstrate that Pyrococcus prefoldin interacts most tightly with its cognate chaperonin, and vice versa, suggesting species specificity in the interaction. Using truncation mutants, we uncovered by kinetic analyses that this interaction is multivalent in nature, consistent with multiple binding sites between the two chaperones. We present evidence that both N-and C-terminal regions of the prefoldin ␤ subunit are important for molecular chaperone activity and for the interaction with a chaperonin. Our data are consistent with substrate and chaperonin binding sites on prefoldin that are different but in close proximity, which suggests a possible handover mechanism of prefoldin substrates to the chaperonin.Molecular chaperones are ubiquitous proteins that are required for the correct folding, assembly, transport, and degradation of proteins within the cell (1). One class of chaperones, termed chaperonins, are seven-to nine-membered double ring complexes of 800 -1000 kDa that capture non-native proteins in their central cavity to promote correct folding in an ATP-dependent manner (2-5). They are classified into two groups, group I found in bacteria and organelles of eukaryotes, and group II in archaea and in the cytoplasm of eukaryotes (6, 7). The bacterial group I chaperonin, GroEL, is a cylinder-shaped tetradecamer that is capped by the heptameric co-chaperone, GroES (8 -10). In contrast, the group II chaperonin exists as an eight-or nine-rotationally symmetric double ring in a toroidal structure composed of homologous subunits of about 60 kDa and functions independently of a co-chaperone corresponding to GroES (11,12). The crystal structures of the group II chaperonins from the acidothermophilic archaeum Thermoplasma acidophilum and the hyperthermophilic archaeum Thermococcus sp. strain KS-1 suggest that the long helical protrusions located at the opening of their cavities, in their apical domains, play the equivalent role of GroES as a built-in lid of the cavity (13-15). The archaeal chaperonin takes an open conformation in the nucleotide-free or ADP-bound states and changes to a closed conformation upon binding ATP (16,17). Interestingly, closure of the built-in lid of CCT, 1 the eukaryotic cytosol group II chaperonin, is induced not by the binding but by the hydrolysis of ATP (18).The group II chaperonins cooperate with a co-chaperone, prefoldin/GimC (19 -21). Prefoldin (PFD) has been shown to participate in the maturation of actin and members of the tubulin family by transferring them in the incomplete...

Section: Methodsmentioning

confidence: 99%

Kinetics and Binding Sites for Interaction of the Prefoldin with a Group II Chaperonin

Okochi

Nomura

Zako

et al. 2004

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“…strain KS-1. The natural chaperonin derived from T. KS-1 is a hetero-oligomer of ␣ and ␤ subunits, and the composition varies with the growth temperature (14). In addition, each subunit forms a double ring homo-oligomer capable of ATP-dependent protein folding in vitro (10).…”

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confidence: 99%

Role of the Helical Protrusion in the Conformational Change and Molecular Chaperone Activity of the Archaeal Group II Chaperonin

Iizuka

Inobe

et al. 2004

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To elucidate the exact role of the helical protrusion of a group II chaperonin in its molecular chaperone function, three deletion mutants of the chaperonin from a hyperthermophilic archaeum (Thermococcus sp. strain KS-1) lacking one-third, two-thirds, and the whole of the helical protrusion were constructed. The helical protrusion is thought to be substituted for the co-chaperonin GroES of a group I chaperonin and to be important for binding to unfolded proteins. Protease sensitivity assays and small angle x-ray scattering experiments were performed to demonstrate the conformation change of the wild type protein and the deletion mutants by adenine nucleotides. Whereas the binding of ATP to the wild type protein induced a structural transition corresponding to the closure of the built-in lid, it did not cause significant structural changes in deletion mutants. Although the mutants effectively protected proteins from thermal aggregation, ATP-dependent protein folding ability was remarkably diminished. We conclude that the helical protrusion is not necessarily important for binding to unfolded proteins, but its ATP-dependent conformational change mediates folding of captured unfolded proteins.Protein folding is assisted by a number of molecular chaperones in vivo. The chaperonins, a ubiquitous class of molecular chaperones, form double ring complexes that mediate the folding of nascent and denatured proteins in an ATP-dependent manner (1, 2). There are two distinct groups of chaperonins: (i) group I chaperonins of eubacteria and endosymbiotic organelles (mitochondria and chloroplasts); and (ii) group II chaperonins of archaea (known as thermosome) and the eukaryotic cytoplasm (known as CCT 1 or TRiC) (3, 4). Despite the relatively low sequence homology between the two groups, their domain architectures are very similar. Both of them are folded into three domains. The equatorial domain contains the ATP binding site and is involved in intra-and inter-ring contacts. The apical domain is involved in the binding to substrate proteins. The intermediate domain connects the equatorial and apical domains of each subunit and transfers the ATP-induced conformational changes from the equatorial to the apical domain (5, 6).The most striking structural difference between them is the lid of the central cavity of the chaperonin complex. The co-chaperonin GroES/cpn-10 serves as a lid for group I chaperonins in a heptameric dome-like structure. GroES/cpn-10 interacts with one or both GroEL rings in an ATP-regulated fashion, thereby sealing the cavity from the outside. It is demonstrated that a polypeptide of up to ϳ57 kDa is able to fold within the GroESsealed cavity (7). On the other hand, there is no GroES-like co-chaperonin for group II chaperonins. Instead, group II chaperonins have a built-in lid, which is composed of an extension of the apical domain called the helical protrusion. The helical protrusion is thought to play the equivalent role of GroES, sealing off the central cavity of the chaperonin complex (6, 8 -12). Furthe...

“…strain KS-1 (T. KS-1), is one of the most studied group II chaperonins (17)(18)(19)(20)(21)(22)(23). T. KS-1 chaperonin is composed of two highly homologous subunits, ␣ and ␤.…”

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confidence: 99%

“…T. KS-1 chaperonin is composed of two highly homologous subunits, ␣ and ␤. Although the natural chaperonin isolated from T. KS-1 is a hetero-oligomer, each of the recombinant ␣ and ␤ subunits forms a double-ring homo-oligomer and functions as a chaperonin in vitro (17)(18)(19). Recent experimental results using ␤-chaperonin and natural chaperonin suggest that the protein folding occurs within the cis-cavity (21).…”

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confidence: 99%

ATP Binding Is Critical for the Conformational Change from an Open to Closed State in Archaeal Group II Chaperonin

Iizuka

Yoshida

Shomura

et al. 2003

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Group II chaperonins, found in archaea and in eukaryotic cytosol, do not have a co-chaperonin corresponding to GroES. Instead, it is suggested that the helical protrusion extending from the apical domain acts as a built-in lid for the central cavity and that the opening and closing of the lid is regulated by ATP binding and hydrolysis. However, details of this conformational change remain unclear. To investigate the conformational change associated with the ATP-driven cycle, we conducted protease sensitivity analyses and tryptophan fluorescence spectroscopy of ␣-chaperonin from a hyperthermophilic archaeum, Thermococcus strain KS-1. In the nucleotide-free or ADP-bound state, the chaperonin, especially in the helical protrusion region, was highly sensitive to proteases. Addition of ATP and ammonium sulfate induced the transition to the relatively protease-resistant form. The fluorescence intensity of the tryptophan residue introduced at the tip of the helical protrusion was enhanced by the presence of ATP or ammonium sulfate. We conclude that ATP binding induces the conformational change from the lid-open to lid-closed form in archaeal group II chaperonin.Chaperonins, one of the principal molecular chaperones, capture non-native proteins and promote folding in vivo and in vitro in an ATP-dependent manner. They form large cylindrical complexes composed of two stacked rings of 7-9 subunits, each about 60 kDa in size. There is a large cavity in the center of the complex, where unfolded proteins may be encapsulated and undergo productive folding (1, 2). Based on protein sequence similarity and structural features, chaperonins are grouped into two subfamilies (3, 4). Group I chaperonins are found in bacteria and eukaryotic organelles, mitochondria and chloroplasts. The Escherichia coli chaperonin, GroEL, is the most extensively characterized member of the chaperonins. GroEL facilitates protein folding with a cofactor termed GroES in an ATP-dependent manner. GroES has two functions; it acts as a lid to cap the cavity of GroEL, and then induces the release of bound substrate protein into the GroEL-GroES cavity where it can undergo folding (5).The group II chaperonins are found in archaea (as thermosome) and in the cytosol of eukaryotic cells (as CCT or TRiC). 1Group II chaperonins do not have a co-chaperonin corresponding to GroES. The crystal structure of the group II chaperonin from an acidothermophilic archaeum, Thermoplasma acidophilum, suggested that the function of GroES is replaced by long helical protrusions from the apical domain (6, 7). These protrusions are thought to function as a "built-in lid" for the central cavity. Presumably, ATP binding drives group II chaperonins from the lid-open, substrate binding conformation, into the lid-closed conformation, where substrate folds within the central cavity (6 -9). However, the exact relationship between the nucleotide-bound state and the conformation is still controversial in group II chaperonins. Szpikowska et al. (10) reported that CCT is more resistant to tryp...