Chaperonin cofactors, Cpn10 and Cpn20, of green algae and plants function as hetero-oligomeric ring complexes

The chloroplast chaperonin system of plants and green algae is a curiosity as both the chaperonin cage and its lid are encoded by multiple genes, in contrast to the single genes encoding the two components of the bacterial and mitochondrial systems. In the green alga Chlamydomonas reinhardtii (Cr), three genes encode chaperonin cofactors, with cpn10 encoding a single ～10-kDa domain and cpn20 and cpn23 encoding tandem cpn10 domains. Here, we characterized the functional interaction of these proteins with the Escherichia coli chaperonin, GroEL, which normally cooperates with GroES, a heptamer of ～10-kDa subunits. The C. reinhardtii cofactor proteins alone were all unable to assist GroEL-mediated refolding of bacterial ribulose-bisphosphate carboxylase/oxygenase but gained this ability when CrCpn20 and/or CrCpn23 was combined with CrCpn10. Native mass spectrometry indicated the formation of hetero-oligomeric species, consisting of seven ～10-kDa domains. The cofactor "heptamers" interacted with GroEL and encapsulated substrate protein in a nucleotide-dependent manner. Different hetero-oligomer arrangements, generated by constructing cofactor concatamers, indicated a preferential heptamer configuration for the functional CrCpn10-CrCpn23 complex. Formation of heptamer Cpn10/Cpn20 hetero-oligomers was also observed with the Arabidopsis thaliana (At) cofactors, which functioned with the chloroplast chaperonin, AtCpn60α(7)β(7). It appears that hetero-oligomer formation occurs more generally for chloroplast chaperonin cofactors, perhaps adapting the chaperonin system for the folding of specific client proteins.     

A systematic survey of in vivo obligate chaperonin‐dependent substrates

Chaperonins are absolutely required for the folding of a subset of proteins in the cell. An earlier proteome‐wide analysis of Escherichia coli chaperonin GroEL/GroES (GroE) interactors predicted obligate chaperonin substrates, which were termed Class III substrates. However, the requirement of chaperonins for in vivo folding has not been fully examined. Here, we comprehensively assessed the chaperonin requirement using a conditional GroE expression strain, and concluded that only ～60% of Class III substrates are bona fide obligate GroE substrates in vivo. The in vivo obligate substrates, combined with the newly identified obligate substrates, were termed Class IV substrates. Class IV substrates are restricted to proteins with molecular weights that could be encapsulated in the chaperonin cavity, are enriched in alanine/glycine residues, and have a strong structural preference for aggregation‐prone folds. Notably, ～70% of the Class IV substrates appear to be metabolic enzymes, supporting a hypothetical role of GroE in enzyme evolution.     

Arabidopsis chloroplast chaperonin 10 is a calmodulin-binding protein

Calcium regulates diverse cellular activities in plants through the action of calmodulin (CaM). By using (35)S-labeled CaM to screen an Arabidopsis seedling cDNA expression library, a cDNA designated as AtCh-CPN10 (Arabidopsis thaliana chloroplast chaperonin 10) was cloned. Chloroplast CPN10, a nuclear-encoded protein, is a functional homolog of E. coli GroES. It is believed that CPN60 and CPN10 are involved in the assembly of Rubisco, a key enzyme involved in the photosynthetic pathway. Northern analysis revealed that AtCh-CPN10 is highly expressed in green tissues. The recombinant AtCh-CPN10 binds to CaM in a calcium-dependent manner. Deletion mutants revealed that there is only one CaM-binding site in the last 31 amino acids of the AtCh-CPN10 at the C-terminal end. The CaM-binding region in AtCh-CPN10 has higher homology to other chloroplast CPN10s in comparison to GroES and mitochondrial CPN10s, suggesting that CaM may only bind to chloroplast CPN10s. Furthermore, the results also suggest that the calcium/CaM messenger system is involved in regulating Rubisco assembly in the chloroplast, thereby influencing photosynthesis.     

BeF(x) stops the chaperonin cycle of GroEL-GroES and generates a complex with double folding chambers

Coupling with ATP hydrolysis and cooperating with GroES, the double ring chaperonin GroEL assists the folding of other proteins. Here we report novel GroEL-GroES complexes formed in fluoroberyllate (BeF(x)) that can mimic the phosphate part of the enzyme-bound nucleotides. In ATP, BeF(x) stops the functional turnover of GroEL by preventing GroES release and produces a symmetric 1:2 GroEL-GroES complex in which both GroEL rings contain ADP.BeF(x) and an encapsulated substrate protein. In ADP, the substrate protein-loaded GroEL cannot bind GroES. In ADP plus BeF(x), however, it can bind GroES to form a stable 1:1 GroEL-GroES complex in which one of GroEL rings contains ADP.BeF(x) and an encapsulated substrate protein. This 1:1 GroEL-GroES complex is converted into the symmetric 1:2 GroEL-GroES complex when GroES is supplied in ATP plus BeF(x). Thus, BeF(x) stabilizes two GroEL-GroES complexes; one with a single folding chamber and the other with double folding chambers. These results shed light on the intermediate ADP.P(i) nucleotide states in the functional cycle of GroEL.     

Co‐chaperonin GroES as a modulator of proteasomal activity

The proteasome has a crucial part in the degradation of normal, damaged, mutant or misfolded proteins within both the ubiquitin ATP‐dependent and the ubiquitin ATP‐independent pathways. Proteasome‐mediated proteolysis is modulated by diverse factors, and in this regard, chaperonins have been attracting great interest. The investigation on the role of a co‐chaperonin, namely GroES, in the modulation of proteasomal activity was the focus of this work. Our study reports on an analytical approach based on combined fluorimetric, chromatographic (applied to the enzymatic activity evaluation), surface plasmon resonance techniques and molecular modelling, addressed to the assessment and characterization of the interaction. Globally, we described a high affinity interaction between GroES and two different 20 S (immuno‐ and constitutive) proteasomes, uncovering new scenarios on their possible physio‐pathological role, specifically on the ability of proteasomes to interact both with unfolding and folding‐ assisting macromolecules. Copyright © 2008 John Wiley & Sons, Ltd.     

A protein with an inactive pterin‐4a‐carbinolamine dehydratase domain is required for Rubisco biogenesis in plants

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) plays a critical role in sustaining life by catalysis of carbon fixation in the Calvin–Benson pathway. Incomplete knowledge of the assembly pathway of chloroplast Rubisco has hampered efforts to fully delineate the enzyme's properties, or seek improved catalytic characteristics via directed evolution. Here we report that a Mu transposon insertion in the Zea mays (maize) gene encoding a chloroplast dimerization co‐factor of hepatocyte nuclear factor 1 (DCoH)/pterin‐4α‐carbinolamine dehydratases (PCD)‐like protein is the causative mutation in a seedling‐lethal, Rubisco‐deficient mutant named Rubisco accumulation factor 2 (raf2‐1). In raf2 mutants newly synthesized Rubisco large subunit accumulates in a high‐molecular weight complex, the formation of which requires a specific chaperonin 60‐kDa isoform. Analogous observations had been made previously with maize mutants lacking the Rubisco biogenesis proteins RAF1 and BSD2. Chemical cross‐linking of maize leaves followed by immunoprecipitation with antibodies to RAF2, RAF1 or BSD2 demonstrated co‐immunoprecipitation of each with Rubisco small subunit, and to a lesser extent, co‐immunoprecipitation with Rubisco large subunit. We propose that RAF2, RAF1 and BSD2 form transient complexes with the Rubisco small subunit, which in turn assembles with the large subunit as it is released from chaperonins.     

Electron Microscopy of Bacillus subtilis GroESL Chaperonin and Interaction with the Bacteriophage φ29 Head-Tail Connector

The Bacillus subtilis GroESL chaperonin was isolated by sucrose density gradient centrifugation and the constituent GroES and GroEL moieties were purified by electrophoresis in agarose. Electron microscopic images of negatively stained GroEL and GroES oligomers and GroESL complexes were averaged using a reference-free alignment method. The GroEL and GroES particles had the sevenfold symmetry characteristic of their Escherichia coli counterparts. GroESL complexes, reconstituted efficiently in vitro from GroEL and GroES in the absence of added ADP or ATP, had the characteristic bullet- and football-like shapes in side view. Purified bacteriophage φ29 head-tail connectors having a mass in excess of 0.4 MDa were shown to bind to GroESL at the end opposite to the GroES. The same GroESL-connector complexes were isolated from phage-infected cells in which capsid assembly was blocked, and thus the complex may have functional significance in φ29 morphogenesis.     

Coexistence of group I and group II chaperonins in the archaeon Methanosarcina mazei

Two distantly related classes of cylindrical chaperonin complexes assist in the folding of newly synthesized and stress-denatured proteins in an ATP-dependent manner. Group I chaperonins are thought to be restricted to the cytosol of bacteria and to mitochondria and chloroplasts, whereas the group II chaperonins are found in the archaeal and eukaryotic cytosol. Here we show that members of the archaeal genus Methanosarcina co-express both the complete group I (GroEL/GroES) and group II (thermosome/prefoldin) chaperonin systems in their cytosol. These mesophilic archaea have acquired between 20 and 35% of their genes by lateral gene transfer from bacteria. In Methanosarcina mazei G?1, both chaperonins are similarly abundant and are moderately induced under heat stress. The M. mazei GroEL/GroES proteins have the structural features of their bacterial counterparts. The thermosome contains three paralogous subunits, alpha, beta, and gamma, which assemble preferentially at a molar ratio of 2:1:1. As shown in vitro, the assembly reaction is dependent on ATP/Mg2+ or ADP/Mg2+ and the regulatory role of the beta subunit. The co-existence of both chaperonin systems in the same cellular compartment suggests the Methanosarcina species as useful model systems in studying the differential substrate specificity of the group I and II chaperonins and in elucidating how newly synthesized proteins are sorted from the ribosome to the proper chaperonin for folding.     

An expanded protein folding cage in the GroEL-gp31 complex

Bacteriophage T4 produces a GroES analogue, gp31, which cooperates with the Escherichia coli GroEL to fold its major coat protein gp23. We have used cryo-electron microscopy and image processing to obtain three-dimensional structures of the E.coli chaperonin GroEL complexed with gp31, in the presence of both ATP and ADP. The GroEL-gp31-ADP map has a resolution of 8.2 A, which allows accurate fitting of the GroEL and gp31 crystal structures. Comparison of this fitted structure with that of the GroEL-GroES-ADP structure previously determined by cryo-electron microscopy shows that the folding cage is expanded. The enlarged volume for folding is consistent with the size of the bacteriophage coat protein gp23, which is the major substrate of GroEL-gp31 chaperonin complex. At 56 kDa, gp23 is close to the maximum size limit of a polypeptide that is thought to fit inside the GroEL-GroES folding cage.     

Identification of Elements That Dictate the Specificity of Mitochondrial Hsp60 for Its Co-Chaperonin

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.     

Requirement for GroEL/GroES-dependent protein folding under nonpermissive conditions of macromolecular crowding

Macromolecular crowding is a critical parameter affecting the efficiency of cellular protein folding. Here we show that the proteins dihydrofolate reductase, enolase, and green fluorescent protein, which can fold spontaneously in diluted buffer, lose this ability in a crowded environment. Instead, they accumulate as soluble, protease-sensitive non-native species. Their folding becomes dependent on the complete GroEL/GroES chaperonin system and is not affected by trap-GroEL, indicating that folding has to occur in the chaperonin cavity with release of nativelike proteins into the bulk solution. In addition, we demonstrate that efficient folding in the chaperonin cavity requires ATP hydrolysis, as formation of ternary GroEL/GroES complexes with substrate proteins in the presence of ADP results only in very inefficient reactivation. However, protein refolding reactions using ADP-fluoroaluminate complexes, or single-ring GroEL and GroES under conditions where only a single round of ATP hydrolysis occurs, yield large amounts of refolded enzymes. Thus, the mode of initial ternary complex formation appears to be critical for subsequent productive release of substrate into the cavity under certain crowding conditions, and is only efficient when triggered by ATP hydrolysis. Our data indicate that stringent conditions of crowding can impart a stronger dependence of folding proteins on the assistance by chaperonins.     

Analysis of the interaction mode between hyperthermophilic archaeal group II chaperonin and prefoldin using a platform of chaperonin oligomers of various subunit arrangements

Prefoldin is a co-chaperone that captures an unfolded protein substrate and transfers it to the group II chaperonin for completion of protein folding. Group II chaperonin of a hyperthermophilic archaeon, Thermococcus strain KS-1, interacts and cooperates with archaeal prefoldins. Although the interaction sites within chaperonin and prefoldin have been analyzed, the binding mode between jellyfish-like hexameric prefoldin and the double octameric ring group II chaperonin remains unclear. As prefoldin binds the chaperonin β subunit more strongly than the α subunit, we analyzed the binding mode between prefoldin and chaperonin in the context of Thermococcus group II chaperonin complexes of various subunit compositions and arrangements. The oligomers exhibited various affinities for prefoldins according to the number and order of subunits. Binding affinity increased with the number of Cpnβ subunits. Interestingly, chaperonin complexes containing two β subunits adjacently exhibited stronger affinities than other chaperonin complexes containing the same number of β subunits. The result suggests that all four β tentacles of prefoldin interact with the helical protrusions of CPN in the PFD–CPN complex as the previously proposed model that two adjacent PFD β subunits seem to interact with two CPN adjacent subunits.     

On the role of symmetrical and asymmetrical chaperonin complexes in assisted protein folding.

The cylindrical chaperonin GroEL of E. coli and its ring-shaped cofactor GroES cooperate in mediating the ATP-dependent folding of a wide range of polypeptides in vivo and in vitro. By binding to the ends of the GroEL cylinder, GroES displaces GroEL-bound polypeptide into an enclosed folding cage, thereby preventing protein aggregation during folding. The dynamic interaction of GroEL and GroES is regulated by the GroEL ATPase and involves the formation of asymmetrical GroEL:GroES1 and symmetrical GroEL: GroES2 complexes. The proposed role of the symmetrical complex as a catalytic intermediate of the chaperonin mechanism has been controversial. It has also been suggested that the formation of GroEL:GroES2 complexes allows the folding of two polypeptide molecules per GroEL reaction cycle, one in each ring of GroEL. By making use of a procedure to stabilize chaperonin complexes by rapid crosslinking for subsequent analysis by native PAGE, we have quantified the occurrence of GroEL:GroES1 and GroEL:GroES2 complexes in active refolding reactions under a variety of conditions using mitochondrial malate dehydrogenase (mMDH) as a substrate. Our results show that the symmetrical complexes are neither required for chaperonin function nor does their presence significantly increase the rate of mMDH refolding. In contrast, chaperonin-assisted folding is strictly dependent on the formation of asymmetrical GroEL:GroES1 complexes. These findings support the view that GroEL:GroES2 complexes have no essential role in the chaperonin mechanism.     

Synthesis and Biological Evaluation of Gramicidin S‐Inspired Cyclic Mixed α/β‐Peptides

Via a Mannich reaction involving a dibenzyliminium species and the titanium enolates of Evans' chiral acylated oxazolidinones the β²‐amino acids (R)‐ and (S)‐Fmoc‐β²homovaline and (R)‐Fmoc‐β²homoleucine are synthesized. These building blocks were used, in combination with commercially available α‐ and β³‐amino acids, for the synthesis of the cyclo‐(αβ³αβ²α)₂ peptide 2 and the cyclo‐(αβ²αβ³α)₂ peptides 3 – 5 . The peptides 2 – 5 were screened for their ability to inhibit a small panel of Gram‐negative and Gram‐positive bacterial strains.     

Discrimination of ATP,ADP, and AMPPNP by chaperonin GroEL: hexokinase treatment revealed the exclusive role of ATP

The double ring chaperonin GroEL binds unfolded protein, ATP, and GroES to the same ring, generating the cis ternary complex in which folding occurs within the cavity capped by GroES (cis folding). The functional role of ATP, however, remains unclear since several reports have indicated that ADP and AMPPNP (5'-adenylyl-beta,gamma-imidodiphosphate) are also able to support the formation of the cis ternary complex and the cis folding. To minimize the effect of contaminated ATP and adenylate kinase, we have included hexokinase plus glucose in the reaction mixtures and obtained new results. In ADP and AMPPNP, GroES bound quickly to GroEL but bound very slowly to the GroEL loaded with unfolded rhodanese or malate dehydrogenase. ADP was unable to support the formation of cis ternary complex and cis folding. AMPPNP supported cis folding of malate dehydrogenase to some extent but not cis folding of rhodanese. In the absence of hexokinase, apparent cis folding of rhodanese and malate dehydrogenase was observed in ADP and AMPPNP. Thus, the exclusive role of ATP in generation of the cis ternary complex is now evident.     

Thermodynamic Characterization of the Interaction between Prefoldin and Group II Chaperonin

Prefoldin (PFD) is a hexameric chaperone that captures a protein substrate and transfers it to a group II chaperonin (CPN) to complete protein folding. We have studied the interaction between PFD and CPN using those from a hyperthermophilic archaeon, Thermococcus strain KS-1 (T. KS-1). In this study, we determined the crystal structure of the T. KS-1 PFDβ2 subunit and characterized the interactions between T. KS-1 CPNs (CPNα and CPNβ) and T. KS-1 PFDs (PFDα1-β1 and PFDα2-β2). As predicted from its amino acid sequence, the PFDβ2 subunit conforms to a structure similar to those of the PFDβ1 subunit and the Pyrococcus horikoshii OT3 PFDβ subunit, with the exception of the tip of its coiled-coil domain, which is thought to be the CPN interaction site. The interactions between T. KS-1 CPNs and PFDs (CPNα and PFDα1-β1; CPNα and PFDα2-β2; CPNβ and PFDα1-β1; and CPNβ and PFDα2-β2) were analyzed using the Biacore T100 system at various temperatures ranging from 20 to 45 ºC. The affinities between PFDs and CPNs increased with an increase in temperature. The thermodynamic parameters calculated from association constants showed that the interaction between PFD and CPN is entropy driven. Among the four combinations of PFD-CPN interactions, the entropy difference in binding between CPNβ and PFDα2-β2 was the largest, and affinity significantly increased at higher temperatures. Considering that expression of PFDα2-β2 and CPNβ subunit is induced upon heat shock, our results suggest that PFDα1-β1 is a general PFD for T. KS-1 CPNs, whereas PFDα2-β2 is specific for CPNβ.     

Dual function of protein confinement in chaperonin-assisted protein folding.

The GroEL/GroES chaperonin system mediates the folding of a range of newly synthesized polypeptides in the bacterial cytosol. Using a rapid biotin-streptavidin-based inhibition of chaperonin function, we show that the cage formed by GroEL and its cofactor GroES can have a dual role in promoting folding. First, enclosure of nonnative protein in the GroEL:GroES complex is essential for folding to proceed unimpaired by aggregation. Second, folding inside the cage can be significantly faster than folding in free solution, independently of ATP-driven cycles of GroES binding and release. This suggests that confinement of unfolded protein in the narrow hydrophilic space of the chaperonin cage smoothes the energy landscape for the folding of some proteins, increasing the flux of folding intermediates toward the native state.     

"Half of the sites" binding of D-glyceraldehyde-3-phosphate dehydrogenase folding intermediate with GroEL

Two D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) folding intermediate subunits bind with chaperonin 60 (GroEL) to form a stable complex, which can no longer bind with additional GAPDH intermediate subunits, but does bind with one more lysozyme folding intermediate or one chaperonin 10 (GroES) molecule, suggesting that the two GAPDH subunits bind at one end of the GroEL molecule displaying a "half of the sites" binding profile. For lysozyme, GroEL binds with either one or two folding intermediates to form a stable 1:1 or 1:2 complex with one substrate on each end of the GroEL double ring for the latter. The 1:1 complex of GroEL.GroES binds with one lysozyme or one dimeric GAPDH folding intermediate to form a stable ternary complex. Both complexes of GroEL.lysozyme1 and GroEL.GAPDH2 bind with one GroES molecule only at the other end of the GroEL molecule forming a trans ternary complex. According to the stoichiometry of GroEL binding with the GAPDH folding intermediate and the formation of ternary complexes containing GroEL.GAPDH2, it is suggested that the folding intermediate of GAPDH binds, very likely in the dimeric form, with GroEL at one end only.     

Mechanism of chaperonin action: GroES binding and release can drive GroEL-mediated protein folding in the absence of ATP hydrolysis.

As a basic principle, assisted protein folding by GroEL has been proposed to involve the disruption of misfolded protein structures through ATP hydrolysis and interaction with the cofactor GroES. Here, we describe chaperonin subreactions that prompt a re-examination of this view. We find that GroEL-bound substrate polypeptide can induce GroES cycling on and off GroEL in the presence of ADP. This mechanism promotes efficient folding of the model protein rhodanese, although at a slower rate than in the presence of ATP. Folding occurs when GroES displaces the bound protein into the sequestered volume of the GroEL cavity. Resulting native protein leaves GroEL upon GroES release. A single-ring variant of GroEL is also fully functional in supporting this reaction cycle. We conclude that neither the energy of ATP hydrolysis nor the allosteric coupling of the two GroEL rings is directly required for GroEL/GroES-mediated protein folding. The minimal mechanism of the reaction is the binding and release of GroES to a polypeptide-containing ring of GroEL, thereby closing and opening the GroEL folding cage. The role of ATP hydrolysis is mainly to induce conformational changes in GroEL that result in GroES cycling at a physiologically relevant rate.