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Tadashi Maruyama - One of the best experts on this subject based on the ideXlab platform.
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Natural Chaperonin of the hyperthermophilic archaeum, Thermococcus strain KS-1: a hetero-oligomeric Chaperonin with variable subunit composition
Molecular Microbiology, 2004Co-Authors: Takao Yoshida, Masafumi Yohda, Akira Ideno, Shuichi Hiyamuta, Tadashi MaruyamaAbstract:To study the difference in expression of the Chaperonin α- and β-subunits in Thermococcus strain KS-1 (T. KS-1), we measured their intracellular contents at various growth temperatures using subunit-specific antibodies. The β-subunit was significantly more abundant with increasing temperature (maximum at 93°C), whereas the α-subunit was not. Native PAGE with Western blot analysis indicated that the natural Chaperonins in the crude extracts of T. KS-1 cells grown between 65°C and 95°C migrate as single bands with different mobility. The recombinant α- and β-subunit homo-oligomers migrated differently from each other and from natural Chaperonins. Immunoprecipitation also showed that the natural Chaperonin was the hetero-oligomer. These results indicate that Chaperonin in T. KS-1 formed a hetero-oligomer with variable subunit composition, and that the β-subunit may be adapted to a higher temperature than the α-subunit. T. KS-1 probably changes its Chaperonin subunit composition to acclimatize to the ambient temperature.
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archaeal group ii Chaperonin mediates protein folding in the cis cavity without a detachable groes like co Chaperonin
Journal of Molecular Biology, 2002Co-Authors: Takao Yoshida, Hideki Taguchi, Masafumi Yohda, Tadashi Maruyama, Rika Kawaguchi, Masasuke Yoshida, Takuo Yasunaga, Takeyuki WakabayashiAbstract:Group II Chaperonins of archaea and eukaryotes are distinct from group I Chaperonins of bacteria. Whereas group I Chaperonins require the co-Chaperonin Cpn-10 or GroES for protein folding, no co-Chaperonin has been known for group II. The protein folding mechanism of group II Chaperonins is not yet clear. To understand this mechanism, we examined protein refolding by the recombinant α or β-subunit Chaperonin homo-oligomer (α16mer and β16mer) from a hyperthermoplilic archaeum, Thermococcus strain KS-1, using a model substrate, green fluorescent protein (GFP). The α16mer and β16mer captured the non-native GFP and promoted its refolding without any co-Chaperonin in an ATP dependent manner. A non-hydrolyzable ATP analog, AMP-PNP, induced the GFP refolding mediated by β16mer but not by the α16mer. A mutant α-subunit Chaperonin homo-oligomer (trap-α) could capture the non-native protein but lacked the ability to refold it. Although trap-α suppressed ATP-dependent refolding of GFP mediated by α16mer or β16mer, it did not affect the AMP-PNP-dependent refolding. This indicated that the GFP refolding mediated by β16mer with AMP-PNP was not accessible to the trap-α. Gel filtration chromatography and a protease protection experiment revealed that this refolded GFP, in the presence of AMP-PNP, was associated with β16mer. After the completion of GFP refolding mediated by β16mer with AMP-PNP, addition of ATP induced an additional refolding of GFP. Furthermore, the β16mer preincubated with AMP-PNP showed the ability to capture the non-native GFP. These suggest that AMP-PNP induced one of two Chaperonin rings (cis-ring) to close and induced protein refolding in this ring, and that the other ring (trans-ring) could capture the unfolded GFP which was refolded by adding ATP. The present data indicate that, in the group II Chaperonin of Thermococcus strain KS-1, the protein folding proceeds in its cis-ring in an ATP-dependent fashion without any co-Chaperonin.
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Chaperonin in a Thermophilic Methanogen, Methanococcus Thermolithotrophicus
New Developments in Marine Biotechnology, 1998Co-Authors: Masahiro Furutani, Toshii Iida, Shigeyuki Yamano, Tadashi MaruyamaAbstract:Molecular chaperones play an important role in the protein foldings in vivo. Chaperonin is a 60 kDa major member of molecular chaperones and has two types, Group I and Group II. The Group I Chaperonin is GroEL-like complex in eubacteria, mitochondria and chloroplasts. Whereas archaeral Chaperonin which is coiled thermosomc and eukaryotic cytosol TCP- I protein belong to Group II (Trent et al., 1991). While a Chaperonin was purified from a hyperthermophilic methanogen, Methanopyrus kandleri (Andra et al., 1996), biochemical and functional characters of Chaperonins in methanogens have remained to be clarified. We report here characteristics of Chaperonin of a thermophilic methanogen, Methanococcus thermolithotrophicus.
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Chaperonin in a Thermophilic Methanogen, Methanococcus Thermolithotrophicus
New Developments in Marine Biotechnology, 1998Co-Authors: Masahiro Furutani, Toshii Iida, Shigeyuki Yamano, Tadashi MaruyamaAbstract:Molecular chaperones play an important role in the protein foldings in vivo. Chaperonin is a 60 kDa major member of molecular chaperones and has two types, Group I and Group II. The Group I Chaperonin is GroEL-like complex in eubacteria, mitochondria and chloroplasts. Whereas archaeral Chaperonin which is coiled thermosomc and eukaryotic cytosol TCP- I protein belong to Group II (Trent et al., 1991). While a Chaperonin was purified from a hyperthermophilic methanogen, Methanopyrus kandleri (Andra et al., 1996), biochemical and functional characters of Chaperonins in methanogens have remained to be clarified. We report here characteristics of Chaperonin of a thermophilic methanogen, Methanococcus thermolithotrophicus.
Judith Frydman - One of the best experts on this subject based on the ideXlab platform.
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An information theoretic framework reveals a tunable allosteric network in group II Chaperonins
Nature Structural & Molecular Biology, 2017Co-Authors: Tom Lopez, Kevin Dalton, Anthony Tomlinson, Vijay Pande, Judith FrydmanAbstract:ATP-dependent allosteric regulation of the ring-shaped group II Chaperonins remains ill defined, in part because their complex oligomeric topology has limited the success of structural techniques in suggesting allosteric determinants. Further, their high sequence conservation has hindered the prediction of allosteric networks using mathematical covariation approaches. Here, we develop an information theoretic strategy that is robust to residue conservation and apply it to group II Chaperonins. We identify a contiguous network of covarying residues that connects all nucleotide-binding pockets within each Chaperonin ring. An interfacial residue between the networks of neighboring subunits controls positive cooperativity by communicating nucleotide occupancy within each ring. Strikingly, Chaperonin allostery is tunable through single mutations at this position. Naturally occurring variants at this position that double the extent of positive cooperativity are less prevalent in nature. We propose that being less cooperative than attainable allows Chaperonins to support robust folding over a wider range of metabolic conditions. Identification of a tunable network of covarying residues within group II Chaperonins suggests how these proteins support robust folding of their clients over a wide range of metabolic conditions.
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an information theoretic framework reveals a tunable allosteric network in group ii Chaperonins
Nature Structural & Molecular Biology, 2017Co-Authors: Tom Lopez, Vijay S Pande, Kevin Dalton, Anthony Tomlinson, Judith FrydmanAbstract:ATP-dependent allosteric regulation of the ring-shaped group II Chaperonins remains ill defined, in part because their complex oligomeric topology has limited the success of structural techniques in suggesting allosteric determinants. Further, their high sequence conservation has hindered the prediction of allosteric networks using mathematical covariation approaches. Here, we develop an information theoretic strategy that is robust to residue conservation and apply it to group II Chaperonins. We identify a contiguous network of covarying residues that connects all nucleotide-binding pockets within each Chaperonin ring. An interfacial residue between the networks of neighboring subunits controls positive cooperativity by communicating nucleotide occupancy within each ring. Strikingly, Chaperonin allostery is tunable through single mutations at this position. Naturally occurring variants at this position that double the extent of positive cooperativity are less prevalent in nature. We propose that being less cooperative than attainable allows Chaperonins to support robust folding over a wider range of metabolic conditions.
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The Mechanism and Function of Group II Chaperonins.
Journal of Molecular Biology, 2015Co-Authors: Tom Lopez, Kevin Dalton, Judith FrydmanAbstract:Protein folding in the cell requires the assistance of enzymes collectively called chaperones. Among these, the Chaperonins are 1-MDa ring-shaped oligomeric complexes that bind unfolded polypeptides and promote their folding within an isolated chamber in an ATP-dependent manner. Group II Chaperonins, found in archaea and eukaryotes, contain a built-in lid that opens and closes over the central chamber. In eukaryotes, the Chaperonin TRiC/CCT is hetero-oligomeric, consisting of two stacked rings of eight paralogous subunits each. TRiC facilitates folding of approximately 10% of the eukaryotic proteome, including many cytoskeletal components and cell cycle regulators. Folding of many cellular substrates of TRiC cannot be assisted by any other chaperone. A complete structural and mechanistic understanding of this highly conserved and essential Chaperonin remains elusive. However, recent work is beginning to shed light on key aspects of Chaperonin function and how their unique properties underlie their contribution to maintaining cellular proteostasis.
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cryo em structure of a group ii Chaperonin in the prehydrolysis atp bound state leading to lid closure
Structure, 2011Co-Authors: Judith Frydman, Nicholai R Douglas, Junjie Zhang, Frank Dimaio, Lukasz A Joachimiak, David Baker, Michael Levitt, Wah ChiuAbstract:Summary Chaperonins are large ATP-driven molecular machines that mediate cellular protein folding. Group II Chaperonins use their "built-in lid" to close their central folding chamber. Here we report the structure of an archaeal group II Chaperonin in its prehydrolysis ATP-bound state at subnanometer resolution using single particle cryo-electron microscopy (cryo-EM). Structural comparison of Mm-cpn in ATP-free, ATP-bound, and ATP-hydrolysis states reveals that ATP binding alone causes the Chaperonin to close slightly with a ∼45° counterclockwise rotation of the apical domain. The subsequent ATP hydrolysis drives each subunit to rock toward the folding chamber and to close the lid completely. These motions are attributable to the local interactions of specific active site residues with the nucleotide, the tight couplings between the apical and intermediate domains within the subunit, and the aligned interactions between two subunits across the rings. This mechanism of structural changes in response to ATP is entirely different from those found in group I Chaperonins.
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crystal structures of a group ii Chaperonin reveal the open and closed states associated with the protein folding cycle
Journal of Biological Chemistry, 2010Co-Authors: Jose Henrique Pereira, Judith Frydman, Corie Y Ralston, Nicholai R Douglas, Daniel W Meyer, Kelly M Knee, Daniel R Goulet, Jonathan King, Paul D AdamsAbstract:Chaperonins are large protein complexes consisting of two stacked multisubunit rings, which open and close in an ATP-dependent manner to create a protected environment for protein folding. Here, we describe the first crystal structure of a group II Chaperonin in an open conformation. We have obtained structures of the archaeal Chaperonin from Methanococcus maripaludis in both a peptide acceptor (open) state and a protein folding (closed) state. In contrast with group I Chaperonins, in which the equatorial domains share a similar conformation between the open and closed states and the largest motions occurs at the intermediate and apical domains, the three domains of the archaeal Chaperonin subunit reorient as a single rigid body. The large rotation observed from the open state to the closed state results in a 65% decrease of the folding chamber volume and creates a highly hydrophilic surface inside the cage. These results suggest a completely distinct closing mechanism in the group II Chaperonins as compared with the group I Chaperonins.
Masafumi Yohda - One of the best experts on this subject based on the ideXlab platform.
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eLS - Thermosome: A Group II Chaperonin of Archaea
eLS, 2016Co-Authors: Yohei Y. Yamamoto, Masafumi YohdaAbstract:Chaperonin, heat shock protein 60, plays an important role in the proteostasis of cytosol. Chaperonins are divided into two groups: group I and group II. Group II Chaperonins exist in eukaryotes and archaea, and these Chaperonins are named CCT/TRiC and Thermosome, respectively. Group II Chaperonins have almost the same structure as group I Chaperonins. The main difference is the existence of a helical protrusion, which constitutes a built-in lid of the cavity. Group II Chaperonin captures an unfolded protein in the cavity in the open conformation and changes to the closed conformation in an ATP-dependent manner, which triggers folding of the captured protein. As Thermosome is relatively stable and simple compared with CCT, the conformational change mechanism and also the interaction with co-chaperone, Prefoldin, have been studied in detail using Thermosome. The conformational change procedure of Thermosome will give insights on protein folding mechanism by group II Chaperonins. Key Concepts Group II Chaperonin is an essential cytosolic molecular chaperone in eukaryotes and archaea. The archaeal group II Chaperonin is named Thermosome. Group II Chaperonin has a built-in lid for the central cavity. As ATP induces a conformational change from the open to the closed conformation, twisting of the ring occurs. Group II Chaperonin cooperates with a co-chaperone, Prefoldin, which captures an unfolded protein and transfers it to the central cavity of the group II Chaperonin. Keywords: Hsp60; chaperone; Chaperonin; Thermosome; conformational change; protein folding; ATPase
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functional characterization of recombinant prefoldin complexes from a hyperthermophilic archaeon thermococcus sp strain ks 1
Journal of Molecular Biology, 2008Co-Authors: Ryo Iizuka, Yuri Sugano, Tadayuki Imanaka, Takao Yoshida, Akashi Ohtaki, Shinsuke Fujiwara, Masafumi YohdaAbstract:Prefoldin is a heterohexameric molecular chaperone complex that is found in the eukaryotic cytosol and also in archaea. It captures a nonnative protein and subsequently delivers it to a group II Chaperonin for proper folding. Archaeal prefoldin is a heterocomplex containing two α subunits and four β subunits with the structure of a double β-barrel assembly, with six long coiled coils protruding from it like a jellyfish with six tentacles. We have studied the protein folding mechanism of group II Chaperonin using those of Thermococcus sp. strain KS-1 (T. KS-1) because they exhibit high protein folding activity in vitro. We have also demonstrated functional cooperation between T. KS-1 Chaperonins and prefoldin from Pyrococcus horikoshii OT3. Recent genome analysis has shown that Thermococcus kodakaraensis KOD1 contains two pairs of prefoldin subunit genes, correlating with the existence of two different Chaperonin subunits. In this study, we characterized four different recombinant prefoldin complexes composed of two pairs of prefoldin subunits (α1, α2, β1, and β2) from T. KS-1. All of them (α1–β1, α2–β1, α1–β2, and α2–β2) exist as α2β4 heterohexamers and can protect several proteins from forming aggregates with different activities. We have also compared the collaborative activity between the prefoldin complexes and the cognate Chaperonins. Prefoldin complexes containing the β1 subunit interacted with the Chaperonins more strongly than those with the β2 subunit. The results suggest that Thermococcus spp. express different prefoldins for different substrates or conditions as Chaperonins.
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Characterization of archaeal group II Chaperonin-ADP-metal fluoride complexes: Implications that group II Chaperonins operate as a "two-stroke engine"
Journal of Biological Chemistry, 2005Co-Authors: Ryo Iizuka, Takao Yoshida, Noriyuki Ishii, Tamotsu Zako, Kazunobu Takahashi, Kosuke Maki, Tomonao Inobe, Kunihiro Kuwajima, Masafumi YohdaAbstract:Group II Chaperonins, found in Archaea and in the eukaryotic cytosol, act independently of a cofactor corresponding to GroES of group I Chaperonins. Instead, the helical protrusion at the tip of the apical domain forms a built-in lid of the central cavity. Although many studies on the lid's conformation have been carried out, the conformation in each step of the ATPase cycle remains obscure. To clarify this issue, we examined the effects of ADP-aluminum fluoride (AlFx) and ADP-beryllium fluoride (BeFx) complexes on alpha-Chaperonin from the hyperthermophilic archaeum, Thermococcus sp. strain KS-1. Biochemical assays, electron microscopic observations, and small angle x-ray scattering measurements demonstrate that alpha-Chaperonin incubated with ADP and BeFx exists in an asymmetric conformation; one ring is open, and the other is closed. The result indicates that alpha-Chaperonin also shares the inherent functional asymmetry of bacterial and eukaryotic cytosolic Chaperonins. Most interestingly, addition of ADP and BeFx induced alpha-Chaperonin to encapsulate unfolded proteins in the closed ring but did not trigger their folding. Moreover, alpha-Chaperonin incubated with ATP and AlFx or BeFx adopted a symmetric closed conformation, and its functional turnover was inhibited. These forms are supposed to be intermediates during the reaction cycle of group II Chaperonins.
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kinetics and binding sites for interaction of the prefoldin with a group ii Chaperonin contiguous non native substrate and Chaperonin binding sites in the archaeal prefoldin
Journal of Biological Chemistry, 2004Co-Authors: Mina Okochi, Ryo Iizuka, Tamotsu Zako, Tomoko Nomura, Takatoshi Arakawa, Hiroshi Ueda, Takashi Funatsu, Michel R. Leroux, Masafumi YohdaAbstract: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 beta sub-unit 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.
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Kinetics and binding sites for interaction of the prefoldin with a group II Chaperonin: contiguous non-native substrate and Chaperonin binding sites in the archaeal prefoldin.
Journal of Biological Chemistry, 2004Co-Authors: Mina Okochi, Ryo Iizuka, Tamotsu Zako, Tomoko Nomura, Takatoshi Arakawa, Hiroshi Ueda, Takashi Funatsu, Michel R. Leroux, Masafumi YohdaAbstract:Abstract 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 β sub-unit 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.
Abdussalam Azem - One of the best experts on this subject based on the ideXlab platform.
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Type I Chaperonins: Mechanism and Beyond
Frontiers Media SA, 2018Co-Authors: Adina Breiman, Abdussalam AzemAbstract:Type I Chaperonins are key players in maintaining the proteome of bacteria and organelles of bacterial origin. They are well known for their crucial role in mediating protein folding. For almost three decades, the molecular mechanism of Chaperonin function has been the subject of intensive research. Still, surprising new mechanistic discoveries are constantly reported. It seems that we are far from having a full understanding of the Chaperonin mode of action. Chaperonins are not simply protein folding machines. They also perform diverse extramitochondrial tasks, mainly related to inflammatory and signal transduction processes. This eBook constitutes ten articles highlighting the latest developments related to the divers functions of Type I Chaperonins. As its title, mechanism and beyond, the collection starts with mechanistic view, continues with extracellular functions and ends with biotechnological applications of Type I Chaperonins
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Identification of Elements That Dictate the Specificity of Mitochondrial Hsp60 for Its Co-Chaperonin
2016Co-Authors: Avital Parnas, Peter A. Lund, Shahar Nisemblat, Celeste Weiss, Galit Levy-rimler, Amir Pri-or, Tsaffrir Zor, Peter Bross, Abdussalam AzemAbstract:Type I Chaperonins (cpn60/Hsp60) are essential proteins that mediate the folding of proteins in bacteria, chloroplast and mitochondria. Despite the high sequence homology among Chaperonins, the mitochondrial Chaperonin system has developed unique properties that distinguish it from the widely-studied bacterial system (GroEL and GroES). The most relevant difference to this study is that mitochondrial Chaperonins are able to refold denatured proteins only with the assistance of the mitochondrial co-Chaperonin. This is in contrast to the bacterial Chaperonin, which is able to function with the help of co-Chaperonin from any source. The goal of our work was to determine structural elements that govern the specificity between Chaperonin and co-Chaperonin pairs using mitochondrial Hsp60 as model system. We used a mutagenesis approach to obtain human mitochondrial Hsp60 mutants that are able to function with the bacterial co-Chaperonin, GroES. We isolated two mutants, a single mutant (E321K) and a double mutant (R264K/E358K) that, together with GroES, were able to rescue an E. coli strain, in which the endogenous Chaperonin system was silenced. Although the mutations are located in the apical domain of the Chaperonin, where the interaction with co-Chaperonin takes place, none of the residues are located in positions that are directly responsible for co-Chaperonin binding. Moreover, while both mutants were able to function with GroES, they showed distinct functional and structural properties. Our results indicate that the phenotype of the E321K mutant is caused mainly by a profound increase in the binding affinity to all co
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crystal structure of the human mitochondrial Chaperonin symmetrical football complex
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Shahar Nisemblat, Avital Parnas, Oren Yaniv, Felix Frolow, Abdussalam AzemAbstract:Human mitochondria harbor a single type I Chaperonin system that is generally thought to function via a unique single-ring intermediate. To date, no crystal structure has been published for any mammalian type I Chaperonin complex. In this study, we describe the crystal structure of a football-shaped, double-ring human mitochondrial Chaperonin complex at 3.15 A, which is a novel intermediate, likely representing the complex in an early stage of dissociation. Interestingly, the mitochondrial Chaperonin was captured in a state that exhibits subunit asymmetry within the rings and nucleotide symmetry between the rings. Moreover, the Chaperonin tetradecamers show a different interring subunit arrangement when compared to GroEL. Our findings suggest that the mitochondrial Chaperonins use a mechanism that is distinct from the mechanism of the well-studied Escherichia coli system.
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crystallization and structure determination of a symmetrical football complex of the mammalian mitochondrial hsp60 hsp10 Chaperonins
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2014Co-Authors: Shahar Nisemblat, Avital Parnas, Abdussalam Azem, Oren Yaniv, Felix FrolowAbstract:The mitochondrial Hsp60–Hsp10 complex assists the folding of various proteins impelled by ATP hydrolysis, similar to the bacterial Chaperonins GroEL and GroES. The near-atomic structural details of the mitochondrial Chaperonins are not known, despite the fact that almost two decades have passed since the structures of the bacterial Chaperonins became available. Here, the crystallization procedure, diffraction experiments and structure determination by molecular replacement of the mammalian mitochondrial Chaperonin HSP60 (E321K mutant) and its co-Chaperonin Hsp10 are reported.
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Identification of elements that dictate the specificity of mitochondrial Hsp60 for its co-Chaperonin.
PLoS ONE, 2012Co-Authors: Avital Parnas, Peter A. Lund, Shahar Nisemblat, Celeste Weiss, Galit Levy-rimler, Amir Pri-or, Tsaffrir Zor, Peter Bross, Abdussalam AzemAbstract:Type I Chaperonins (cpn60/Hsp60) are essential proteins that mediate the folding of proteins in bacteria, chloroplast and mitochondria. Despite the high sequence homology among Chaperonins, the mitochondrial Chaperonin system has developed unique properties that distinguish it from the widely-studied bacterial system (GroEL and GroES). The most relevant difference to this study is that mitochondrial Chaperonins are able to refold denatured proteins only with the assistance of the mitochondrial co-Chaperonin. This is in contrast to the bacterial Chaperonin, which is able to function with the help of co-Chaperonin from any source. The goal of our work was to determine structural elements that govern the specificity between Chaperonin and co-Chaperonin pairs using mitochondrial Hsp60 as model system. We used a mutagenesis approach to obtain human mitochondrial Hsp60 mutants that are able to function with the bacterial co-Chaperonin, GroES. We isolated two mutants, a single mutant (E321K) and a double mutant (R264K/E358K) that, together with GroES, were able to rescue an E. coli strain, in which the endogenous Chaperonin system was silenced. Although the mutations are located in the apical domain of the Chaperonin, where the interaction with co-Chaperonin takes place, none of the residues are located in positions that are directly responsible for co-Chaperonin binding. Moreover, while both mutants were able to function with GroES, they showed distinct functional and structural properties. Our results indicate that the phenotype of the E321K mutant is caused mainly by a profound increase in the binding affinity to all co-Chaperonins, while the phenotype of R264K/E358K is caused by a slight increase in affinity toward co-Chaperonins that is accompanied by an alteration in the allosteric signal transmitted upon nucleotide binding. The latter changes lead to a great increase in affinity for GroES, with only a minor increase in affinity toward the mammalian mitochondrial co-Chaperonin.
Takao Yoshida - One of the best experts on this subject based on the ideXlab platform.
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functional characterization of recombinant prefoldin complexes from a hyperthermophilic archaeon thermococcus sp strain ks 1
Journal of Molecular Biology, 2008Co-Authors: Ryo Iizuka, Yuri Sugano, Tadayuki Imanaka, Takao Yoshida, Akashi Ohtaki, Shinsuke Fujiwara, Masafumi YohdaAbstract:Prefoldin is a heterohexameric molecular chaperone complex that is found in the eukaryotic cytosol and also in archaea. It captures a nonnative protein and subsequently delivers it to a group II Chaperonin for proper folding. Archaeal prefoldin is a heterocomplex containing two α subunits and four β subunits with the structure of a double β-barrel assembly, with six long coiled coils protruding from it like a jellyfish with six tentacles. We have studied the protein folding mechanism of group II Chaperonin using those of Thermococcus sp. strain KS-1 (T. KS-1) because they exhibit high protein folding activity in vitro. We have also demonstrated functional cooperation between T. KS-1 Chaperonins and prefoldin from Pyrococcus horikoshii OT3. Recent genome analysis has shown that Thermococcus kodakaraensis KOD1 contains two pairs of prefoldin subunit genes, correlating with the existence of two different Chaperonin subunits. In this study, we characterized four different recombinant prefoldin complexes composed of two pairs of prefoldin subunits (α1, α2, β1, and β2) from T. KS-1. All of them (α1–β1, α2–β1, α1–β2, and α2–β2) exist as α2β4 heterohexamers and can protect several proteins from forming aggregates with different activities. We have also compared the collaborative activity between the prefoldin complexes and the cognate Chaperonins. Prefoldin complexes containing the β1 subunit interacted with the Chaperonins more strongly than those with the β2 subunit. The results suggest that Thermococcus spp. express different prefoldins for different substrates or conditions as Chaperonins.
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Characterization of archaeal group II Chaperonin-ADP-metal fluoride complexes: Implications that group II Chaperonins operate as a "two-stroke engine"
Journal of Biological Chemistry, 2005Co-Authors: Ryo Iizuka, Takao Yoshida, Noriyuki Ishii, Tamotsu Zako, Kazunobu Takahashi, Kosuke Maki, Tomonao Inobe, Kunihiro Kuwajima, Masafumi YohdaAbstract:Group II Chaperonins, found in Archaea and in the eukaryotic cytosol, act independently of a cofactor corresponding to GroES of group I Chaperonins. Instead, the helical protrusion at the tip of the apical domain forms a built-in lid of the central cavity. Although many studies on the lid's conformation have been carried out, the conformation in each step of the ATPase cycle remains obscure. To clarify this issue, we examined the effects of ADP-aluminum fluoride (AlFx) and ADP-beryllium fluoride (BeFx) complexes on alpha-Chaperonin from the hyperthermophilic archaeum, Thermococcus sp. strain KS-1. Biochemical assays, electron microscopic observations, and small angle x-ray scattering measurements demonstrate that alpha-Chaperonin incubated with ADP and BeFx exists in an asymmetric conformation; one ring is open, and the other is closed. The result indicates that alpha-Chaperonin also shares the inherent functional asymmetry of bacterial and eukaryotic cytosolic Chaperonins. Most interestingly, addition of ADP and BeFx induced alpha-Chaperonin to encapsulate unfolded proteins in the closed ring but did not trigger their folding. Moreover, alpha-Chaperonin incubated with ATP and AlFx or BeFx adopted a symmetric closed conformation, and its functional turnover was inhibited. These forms are supposed to be intermediates during the reaction cycle of group II Chaperonins.
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Natural Chaperonin of the hyperthermophilic archaeum, Thermococcus strain KS-1: a hetero-oligomeric Chaperonin with variable subunit composition
Molecular Microbiology, 2004Co-Authors: Takao Yoshida, Masafumi Yohda, Akira Ideno, Shuichi Hiyamuta, Tadashi MaruyamaAbstract:To study the difference in expression of the Chaperonin α- and β-subunits in Thermococcus strain KS-1 (T. KS-1), we measured their intracellular contents at various growth temperatures using subunit-specific antibodies. The β-subunit was significantly more abundant with increasing temperature (maximum at 93°C), whereas the α-subunit was not. Native PAGE with Western blot analysis indicated that the natural Chaperonins in the crude extracts of T. KS-1 cells grown between 65°C and 95°C migrate as single bands with different mobility. The recombinant α- and β-subunit homo-oligomers migrated differently from each other and from natural Chaperonins. Immunoprecipitation also showed that the natural Chaperonin was the hetero-oligomer. These results indicate that Chaperonin in T. KS-1 formed a hetero-oligomer with variable subunit composition, and that the β-subunit may be adapted to a higher temperature than the α-subunit. T. KS-1 probably changes its Chaperonin subunit composition to acclimatize to the ambient temperature.
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archaeal group ii Chaperonin mediates protein folding in the cis cavity without a detachable groes like co Chaperonin
Journal of Molecular Biology, 2002Co-Authors: Takao Yoshida, Hideki Taguchi, Masafumi Yohda, Tadashi Maruyama, Rika Kawaguchi, Masasuke Yoshida, Takuo Yasunaga, Takeyuki WakabayashiAbstract:Group II Chaperonins of archaea and eukaryotes are distinct from group I Chaperonins of bacteria. Whereas group I Chaperonins require the co-Chaperonin Cpn-10 or GroES for protein folding, no co-Chaperonin has been known for group II. The protein folding mechanism of group II Chaperonins is not yet clear. To understand this mechanism, we examined protein refolding by the recombinant α or β-subunit Chaperonin homo-oligomer (α16mer and β16mer) from a hyperthermoplilic archaeum, Thermococcus strain KS-1, using a model substrate, green fluorescent protein (GFP). The α16mer and β16mer captured the non-native GFP and promoted its refolding without any co-Chaperonin in an ATP dependent manner. A non-hydrolyzable ATP analog, AMP-PNP, induced the GFP refolding mediated by β16mer but not by the α16mer. A mutant α-subunit Chaperonin homo-oligomer (trap-α) could capture the non-native protein but lacked the ability to refold it. Although trap-α suppressed ATP-dependent refolding of GFP mediated by α16mer or β16mer, it did not affect the AMP-PNP-dependent refolding. This indicated that the GFP refolding mediated by β16mer with AMP-PNP was not accessible to the trap-α. Gel filtration chromatography and a protease protection experiment revealed that this refolded GFP, in the presence of AMP-PNP, was associated with β16mer. After the completion of GFP refolding mediated by β16mer with AMP-PNP, addition of ATP induced an additional refolding of GFP. Furthermore, the β16mer preincubated with AMP-PNP showed the ability to capture the non-native GFP. These suggest that AMP-PNP induced one of two Chaperonin rings (cis-ring) to close and induced protein refolding in this ring, and that the other ring (trans-ring) could capture the unfolded GFP which was refolded by adding ATP. The present data indicate that, in the group II Chaperonin of Thermococcus strain KS-1, the protein folding proceeds in its cis-ring in an ATP-dependent fashion without any co-Chaperonin.