Hetero-oligomeric CPN60 resembles highly symmetric group-I chaperonin structure revealed by Cryo-EM.

The chloroplast chaperonin system is indispensable for the biogenesis of Rubisco, the key enzyme in photosynthesis. Using Chlamydomonas reinhardtii as a model system, we found that in vivo the chloroplast chaperonin consists of CPN60α, CPN60ß1 and CPN60ß2 and the co-chaperonin of the three subunits CPN20, CPN11 and CPN23. In Escherichia coli, CPN20 homo-oligomers and all possible other chloroplast co-chaperonin hetero-oligomers are functional, but only that consisting of CPN11/20/23-CPN60αß1ß2 can fully replace GroES/GroEL under stringent stress conditions. Endogenous CPN60 was purified and its stoichiometry was determined to be 6:2:6 for CPN60α:CPN60ß1:CPN60ß2. The cryo-EM structures of endogenous CPN60αß1ß2/ADP and CPN60αß1ß2/co-chaperonin/ADP were solved at resolutions of 4.06 and 3.82 Å, respectively. In both hetero-oligomeric complexes the chaperonin subunits within each ring are highly symmetric. Through hetero-oligomerization, the chloroplast co-chaperonin CPN11/20/23 forms seven GroES-like domains, which symmetrically interact with CPN60αß1ß2. Our structure also reveals an uneven distribution of roof-forming domains in the dome-shaped CPN11/20/23 co-chaperonin and potentially diversified surface properties in the folding cavity of the CPN60αß1ß2 chaperonin that might enable the chloroplast chaperonin system to assist in the folding of specific substrates.

Plant RuBisCo assembly in E. coli with five chloroplast chaperones including BSD2.

Plant RuBisCo, a complex of eight large and eight small subunits, catalyzes the fixation of CO2 in photosynthesis. The low catalytic efficiency of RuBisCo provides strong motivation to reengineer the enzyme with the goal of increasing crop yields. However, genetic manipulation has been hampered by the failure to express plant RuBisCo in a bacterial host. We achieved the functional expression of Arabidopsis thaliana RuBisCo in Escherichia coli by coexpressing multiple chloroplast chaperones. These include the chaperonins Cpn60/Cpn20, RuBisCo accumulation factors 1 and 2, RbcX, and bundle-sheath defective-2 (BSD2). Our structural and functional analysis revealed the role of BSD2 in stabilizing an end-state assembly intermediate of eight RuBisCo large subunits until the small subunits become available. The ability to produce plant RuBisCo recombinantly will facilitate efforts to improve the enzyme through mutagenesis.

Asymmetric functional interaction between chaperonin and its plastidic cofactors.

The specific cochaperonin, chloroplast chaperonin (Cpn)20, consisting of two tandem GroES-like domains, is present abundantly in plant and algal chloroplasts, in addition to Cpn10, which is similar in size to GroES. How Cpn20 oligomers, containing six or eight 10-kDa domains, cooperate with the heptameric ring of chaperonin at the same time as encountering symmetry mismatch is unclear. In the present study, we characterized the functional cooperation of cochaperonins, including two plastidic Cpn20 homo-oligomers from Arabidopsis (AtCpn20) and Chlamydomonas (CrCPN20), and one algal CrCPNs hetero-oligomer, consisting of three cochaperonins, CrCPN11, CrCPN20 and CrCPN23, with two chaperonins, Escherichia coli GroEL and Chlamydomonas CrCPN60. AtCpn20 and CrCPNs were functional for assisting both chaperonins in folding model substrates ribulose bisphosphate carboxylase oxygenase from Rhodospirillum rubrum (RrRubisco) in vitro and complementing GroES function in E. coli. CrCPN20 cooperated only with CrCPN60 (and not GroEL) to refold RrRubisco in vitro and showed differential complementation with the two chaperonins in E. coli. Cochaperonin concatamers, consisting of six to eight covalently linked 10-kDa domains, were functionally similar to their respective native forms. Our results indicate that symmetrical match between chaperonin and cochaperonin is not an absolute requisite for functional cooperation.

The dynamic conformational cycle of the group I chaperonin C-termini revealed via molecular dynamics simulation.

Chaperonins are large ring shaped oligomers that facilitate protein folding by encapsulation within a central cavity. All chaperonins possess flexible C-termini which protrude from the equatorial domain of each subunit into the central cavity. Biochemical evidence suggests that the termini play an important role in the allosteric regulation of the ATPase cycle, in substrate folding and in complex assembly and stability. Despite the tremendous wealth of structural data available for numerous orthologous chaperonins, little structural information is available regarding the residues within the C-terminus. Herein, molecular dynamics simulations are presented which localize the termini throughout the nucleotide cycle of the group I chaperonin, GroE, from Escherichia coli. The simulation results predict that the termini undergo a heretofore unappreciated conformational cycle which is coupled to the nucleotide state of the enzyme. As such, these results have profound implications for the mechanism by which GroE utilizes nucleotide and folds client proteins.

The Cpn10(1) co-chaperonin of A. thaliana functions only as a hetero-oligomer with Cpn20.

The A. thaliana genome encodes five co-chaperonin homologs, three of which are destined to the chloroplast. Two of the proteins, Cpn10(2) and Cpn20, form functional homo-oligomers in vitro. In the current work, we present data on the structure and function of the third A. thaliana co-chaperonin, which exhibits unique properties. We found that purified recombinant Cpn10(1) forms inactive dimers in solution, in contrast to the active heptamers that are formed by canonical Cpn10s. Additionally, our data demonstrate that Cpn10(1) is capable of assembling into active hetero-oligomers together with Cpn20. This finding was reinforced by the formation of active co-chaperonin species upon mixing an inactive Cpn20 mutant with the inactive Cpn10(1). The present study constitutes the first report of a higher plant Cpn10 subunit that is able to function only upon formation of hetero-oligomers with other co-chaperonins.

The complexity of chloroplast chaperonins.

Type I chaperonins are large oligomeric protein ensembles that are involved in the folding and assembly of other proteins. Chloroplast chaperonins and co-chaperonins exist in multiple copies of two distinct isoforms that can combine to form a range of labile oligomeric structures. This complex system increases the potential number of chaperonin substrates and possibilities for regulation. The incorporation of unique subunits into the oligomer can modify substrate specificity. Some subunits are upregulated in response to heat shock and some show organ-specific expression, whereas others possess additional functions that are unrelated to their role in protein folding. Accumulating evidence suggests that specific subunits have distinct roles in biogenesis of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco).

Interaction specificity between the chaperone and proteolytic components of the cyanobacterial Clp protease.

The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. In plant chloroplasts and cyanobacteria, the essential constitutive Clp protease consists of the Hsp100/ClpC chaperone partnering a proteolytic core of catalytic ClpP and noncatalytic ClpR subunits. In the present study, we have examined putative determinants conferring the highly specific association between ClpC and the ClpP3/R core from the model cyanobacterium Synechococcus elongatus. Two conserved sequences in the N-terminus of ClpR (tyrosine and proline motifs) and one in the N-terminus of ClpP3 (MPIG motif) were identified as being crucial for the ClpC-ClpP3/R association. These N-terminal domains also influence the stability of the ClpP3/R core complex itself. A unique C-terminal sequence was also found in plant and cyanobacterial ClpC orthologues just downstream of the P-loop region previously shown in Escherichia coli to be important for Hsp100 association to ClpP. This R motif in Synechococcus ClpC confers specificity for the ClpP3/R core and prevents association with E. coli ClpP; its removal from ClpC reverses this core specificity.

Chaperonins: two rings for folding.

Chaperonins are ubiquitous chaperones found in Eubacteria, eukaryotic organelles (group I), Archaea and the eukaryotic cytosol (group II). They all share a common structure and a basic functional mechanism. Although a large amount of information has been gathered for the simpler group I, much less is known about group II chaperonins. Recent crystallographic and electron microscopy structures have provided new insights into the mechanism of these chaperonins and revealed important differences between group I and II chaperonins, mainly in the molecular rearrangements that take place during the functional cycle. These differences are evident for the most complex chaperonin, the eukaryotic cytosolic CCT, which highlights the uniqueness of this important molecular machine.

Chloroplast ß chaperonins from A. thaliana function with endogenous cpn10 homologs in vitro.

The involvement of type I chaperonins in bacterial and organellar protein folding has been well-documented. In E. coli and mitochondria, these ubiquitous and highly conserved proteins form chaperonin oligomers of identical 60 kDa subunits (cpn60), while in chloroplasts, two distinct cpn60 α and ß subunit types co-exist together. The primary sequence of α and ß subunits is ~50% identical, similar to their respective homologies to the bacterial GroEL. Moreover, the A. thaliana genome contains two α and four ß genes. The functional significance of this variability in plant chaperonin proteins has not yet been elucidated. In order to gain insight into the functional variety of the chloroplast chaperonin family members, we reconstituted ß homo-oligomers from A. thaliana following their expression in bacteria and subjected them to a structure-function analysis. Our results show for the first time, that A. thaliana ß homo-oligomers can function in vitro with authentic chloroplast co-chaperonins (ch-cpn10 and ch-cpn20). We also show that oligomers made up of different ß subunit types have unique properties and different preferences for co-chaperonin partners. We propose that chloroplasts may contain active ß homo-oligomers in addition to hetero-oligomers, possibly reflecting a variety of cellular roles.

On the evolutionary origin of the chaperonins.

An analysis of the apical domain of the Group-I and Group-II chaperonins shows that they have structural similarities to two different protein folds: a "swivel-domain" phosphotransferase and a thioredoxin-like peroxiredoxin. There is no significant sequence similarity that supports either similarity and the degree of similarity based on structure is comparable but weak for both relationships. Based on possible evolutionary transitions, we deduced that a phosphotransferase origin would require both a large insertion and deletion of structure whereas a peroxiredoxin origin requires only a peripheral rearrangement, similar to an internal domain-swap. We postulate that this change could have been triggered by the insertion of a peroxiredoxin into the ATPase domain that led to the modern chaperonin domain arrangement. The peroxidoxin fold is the most highly embellished member of the thioredoxin super-family and the insertion event may have "overloaded" the core, leading to a rearrangement. A peroxiredoxin origin for the domain also provides a functional explanation, as the peroxiredoxins can act as chaperones when they adopt a multimeric ring complex, similar to the chaperonin subunit configuration. In addition, several of the GroEL apical domain hydrophobic residues which interact with the unfolded protein are located in a position that corresponds to the protein substrate binding region of the peroxiredoxin fold. We suggest that the origin of the ur-chaperonin from a thioredoxin/peroxiredoxin fold might also account for the number of thioredoxin-fold containing proteins that interact with chaperonins, such as tubulin and phosducin-like proteins.

Differential substrate specificity of group I and group II chaperonins in the archaeon Methanosarcina mazei.

Chaperonins are macromolecular machines that assist in protein folding. The archaeon Methanosarcina mazei has acquired numerous bacterial genes by horizontal gene transfer. As a result, both the bacterial group I chaperonin, GroEL, and the archaeal group II chaperonin, thermosome, coexist. A proteome-wide analysis of chaperonin interactors was performed to determine the differential substrate specificity of GroEL and thermosome. At least 13% of soluble M. mazei proteins interact with chaperonins, with the two systems having partially overlapping substrate sets. Remarkably, chaperonin selectivity is independent of phylogenetic origin and is determined by distinct structural and biochemical features of proteins. GroEL prefers well-conserved proteins with complex alpha/beta domains. In contrast, thermosome substrates comprise a group of faster-evolving proteins and contain a much wider range of different domain folds, including small all-alpha and all-beta modules, and a greater number of large multidomain proteins. Thus, the group II chaperonins may have facilitated the evolution of the highly complex proteomes characteristic of eukaryotic cells.