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Revisiting the GroEL-GroES reaction cycle via the symmetric intermediate implied by novel aspects of the GroEL(D398A) mutant.

ABSTRACT: The Escherichia coli chaperonin GroEL is a double-ring chaperone that assists in protein folding with the aid of GroES and ATP. It is believed that GroEL alternates the folding-active rings and that the substrate protein (and GroES) can bind to the open trans-ring only after ATP in the cis-ring is hydrolyzed. However, we found that a substrate protein prebound to the trans-ring remained bound during the first ATP cycle, and this substrate was assisted by GroEL-GroES when the second cycle began. Moreover, a slow ATP-hydrolyzing GroEL mutant (D398A) in the ATP-bound form bound a substrate protein and GroES to the trans-ring. The apparent discrepancy with the results from an earlier study (Rye, H. S., Roseman, A. M., Chen, S., Furtak, K., Fenton, W. A., Saibil, H. R., and Horwich, A. L. (1999) Cell 97, 325-338) can be explained by the previously unnoticed fact that the ATP-bound form of the D398A mutant exists as a symmetric 1:2 GroEL-GroES complex (the "football"-shaped complex) and that the substrate protein (and GroES) in the medium is incorporated into the complex only after the slow turnover. In light of these results, the current model of the GroEL-GroES reaction cycle via the asymmetric 1:1 GroEL-GroES complex deserves reexamination.

SUBMITTER: Koike-Takeshita A

PROVIDER: S-EPMC3259786 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Football- and bullet-shaped GroEL-GroES complexes coexist during the reaction cycle.

Project description:GroEL is an Escherichia coli chaperonin that is composed of two heptameric rings stacked back-to-back. GroEL assists protein folding with its cochaperonin GroES in an ATP-dependent manner in vitro and in vivo. However, it is still unclear whether GroES binds to both rings of GroEL simultaneously under physiological conditions. In this study, we monitored the GroEL-GroES interaction in the reaction cycle using fluorescence resonance energy transfer. We found that nearly equivalent amounts of symmetric GroEL-(GroES)(2) (football-shaped) complex and asymmetric GroEL-GroES (bullet-shaped) complex coexist during the functional reaction cycle. We also found that D398A, an ATP hydrolysis defective mutant of GroEL, forms a football-shaped complex with ATP bound to the two rings. Furthermore, we showed that ADP prevents the association of ATP to the trans-ring of GroEL, and as a consequence, the second GroES cannot bind to GroEL. Considering the concentrations of ADP and ATP in E. coli, ADP is expected to have a small effect on the inhibition of GroES binding to the trans-ring of GroEL in vivo. These results suggest that we should reconsider the chaperonin-mediated protein-folding mechanism that involves the football-shaped complex.

| S-EPMC3259762 | biostudies-literature

ATP-triggered ADP release from the asymmetric chaperonin GroEL/GroES/ADP7 is not the rate-limiting step of the GroEL/GroES reaction cycle.

Project description:The GroEL/GroES protein folding chamber is formed and dissociated by ATP binding and hydrolysis. ATP hydrolysis in the GroES-bound (cis) ring gates entry of ATP into the opposite unoccupied trans ring, which allosterically ejects cis ligands. While earlier studies suggested that hydrolysis of cis ATP is the rate-limiting step of the cycle (t1/2 approximately 10 s), a recent study suggested that ADP release from the cis ring may be rate-limiting (t1/2 approximately 15-20 s). Here we have measured ADP release using a coupled enzyme assay and observed a t1/2 for release of <or=4-5 s, indicating that this is not the rate-limiting step of the reaction cycle.

| S-EPMC2849271 | biostudies-literature

Creating the Functional Single-Ring GroEL-GroES Chaperonin Systems via Modulating GroEL-GroES Interaction.

Project description:Chaperonin and cochaperonin, represented by E. coli GroEL and GroES, are essential molecular chaperones for protein folding. The double-ring assembly of GroEL is required to function with GroES, and a single-ring GroEL variant GroELSR forms a stable complex with GroES, arresting the chaperoning reaction cycle. GroES I25 interacts with GroEL; however, mutations of I25 abolish GroES-GroEL interaction due to the seven-fold mutational amplification in heptameric GroES. To weaken GroELSR-GroES interaction in a controlled manner, we used groES 7, a gene linking seven copies of groES, to incorporate I25 mutations in selected GroES modules in GroES7. We generated GroES7 variants with different numbers of GroESI25A or GroESI25D modules and different arrangements of the mutated modules, and biochemically characterized their interactions with GroELSR. GroES7 variants with two mutated modules participated in GroELSR-mediated protein folding in vitro. GroES7 variants with two or three mutated modules collaborated with GroELSR to perform chaperone function in vivo: three GroES7 variants functioned with GroELSR under both normal and heat-shock conditions. Our studies on functional single-ring bacterial chaperonin systems are informative to the single-ring human mitochondrial chaperonin mtHsp60-mtHsp10, and will provide insights into how the double-ring bacterial system has evolved to the single-ring mtHsp60-mtHsp10.

| S-EPMC5575113 | biostudies-literature

Substrate protein dependence of GroEL-GroES interaction cycle revealed by high-speed atomic force microscopy imaging.

Project description:A double-ring-shaped tetradecameric GroEL complex assists proper protein folding in cooperation with the cochaperonin GroES. The dynamic GroEL-GroES interaction reflects the allosteric intra- and inter-ring communications and the chaperonin reaction. Therefore, revealing this dynamic interaction is essential to understanding the allosteric communications and the operation mechanism of GroEL. Nevertheless, how this interaction proceeds in the chaperonin cycle has long been controversial. Here, we directly image the dynamic GroEL-GroES interaction under conditions with and without foldable substrate protein using high-speed atomic force microscopy. Then, the imaging results obtained under these conditions and our previous results in the presence of unfoldable substrate are compared. The molecular movies reveal that the entire reaction pathway is highly complicated but basically identical irrespective of the substrate condition. A prominent (but moderate) difference is in the population distribution of intermediate species: symmetric GroEL : GroES2 and asymmetric GroEL : GroES1 complexes, and GroES-unbound GroEL. This difference is mainly attributed to the longer lifetime of GroEL : GroES1 complexes in the presence of foldable substrate. Moreover, the inter-ring communication, which is the basis for the alternating action of the two rings, occurs at two distinct (GroES association and dissociation) steps in the main reaction pathway, irrespective of the substrate condition.This article is part of a discussion meeting issue 'Allostery and molecular machines'.

| S-EPMC5941175 | biostudies-literature

Triggering protein folding within the GroEL-GroES complex.

Project description:The folding of many proteins depends on the assistance of chaperonins like GroEL and GroES and involves the enclosure of substrate proteins inside an internal cavity that is formed when GroES binds to GroEL in the presence of ATP. Precisely how assembly of the GroEL-GroES complex leads to substrate protein encapsulation and folding remains poorly understood. Here we use a chemically modified mutant of GroEL (EL43Py) to uncouple substrate protein encapsulation from release and folding. Although EL43Py correctly initiates a substrate protein encapsulation reaction, this mutant stalls in an intermediate allosteric state of the GroEL ring, which is essential for both GroES binding and the forced unfolding of the substrate protein. This intermediate conformation of the GroEL ring possesses simultaneously high affinity for both GroES and non-native substrate protein, thus preventing escape of the substrate protein while GroES binding and substrate protein compaction takes place. Strikingly, assembly of the folding-active GroEL-GroES complex appears to involve a strategic delay in ATP hydrolysis that is coupled to disassembly of the old, ADP-bound GroEL-GroES complex on the opposite ring.

| S-EPMC2581556 | biostudies-literature

Allosteric signaling of ATP hydrolysis in GroEL-GroES complexes.

Project description:The double-ring chaperonin GroEL and its lid-like cochaperonin GroES form asymmetric complexes that, in the ATP-bound state, mediate productive folding in a hydrophilic, GroES-encapsulated chamber, the so-called cis cavity. Upon ATP hydrolysis within the cis ring, the asymmetric complex becomes able to accept non-native polypeptides and ATP in the open, trans ring. Here we have examined the structural basis for this allosteric switch in activity by cryo-EM and single-particle image processing. ATP hydrolysis does not change the conformation of the cis ring, but its effects are transmitted through an inter-ring contact and cause domain rotations in the mobile trans ring. These rigid-body movements in the trans ring lead to disruption of its intra-ring contacts, expansion of the entire ring and opening of both the nucleotide pocket and the substrate-binding domains, admitting ATP and new substrate protein.

| S-EPMC2871290 | biostudies-literature

Novel cryo-EM structure of an ADP-bound GroEL-GroES complex.

Project description:The GroEL-GroES chaperonin complex is a bacterial protein folding system, functioning in an ATP-dependent manner. Upon ATP binding and hydrolysis, it undergoes multiple stages linked to substrate protein binding, folding and release. Structural methods helped to reveal several conformational states and provide more information about the chaperonin functional cycle. Here, using cryo-EM we resolved two nucleotide-bound structures of the bullet-shaped GroEL-GroES1 complex at 3.4 Å resolution. The main difference between them is the relative orientation of their apical domains. Both structures contain nucleotides in cis and trans GroEL rings; in contrast to previously reported bullet-shaped complexes where nucleotides were only present in the cis ring. Our results suggest that the bound nucleotides correspond to ADP, and that such a state appears at low ATP:ADP ratios.

| S-EPMC8440773 | biostudies-literature

Markov propagation of allosteric effects in biomolecular systems: application to GroEL-GroES.

Project description:We introduce a novel approach for elucidating the potential pathways of allosteric communication in biomolecular systems. The methodology, based on Markov propagation of 'information' across the structure, permits us to partition the network of interactions into soft clusters distinguished by their coherent stochastics. Probabilistic participation of residues in these clusters defines the communication patterns inherent to the network architecture. Application to bacterial chaperonin complex GroEL-GroES, an allostery-driven structure, identifies residues engaged in intra- and inter-subunit communication, including those acting as hubs and messengers. A number of residues are distinguished by their high potentials to transmit allosteric signals, including Pro33 and Thr90 at the nucleotide-binding site and Glu461 and Arg197 mediating inter- and intra-ring communication, respectively. We propose two most likely pathways of signal transmission, between nucleotide- and GroES-binding sites across the cis and trans rings, which involve several conserved residues. A striking observation is the opposite direction of information flow within cis and trans rings, consistent with negative inter-ring cooperativity. Comparison with collective modes deduced from normal mode analysis reveals the propensity of global hinge regions to act as messengers in the transmission of allosteric signals.

| S-EPMC1681507 | biostudies-literature

Chaperonin GroEL uses asymmetric and symmetric reaction cycles in response to the concentration of non-native substrate proteins.

Project description:The Escherichia coli chaperonin GroEL is an essential molecular chaperone that mediates protein folding in association with its cofactor, GroES. It is widely accepted that GroEL alternates the GroES-sealed folding-active rings during the reaction cycle. In other words, an asymmetric GroEL-GroES complex is formed during the cycle, whereas a symmetric GroEL-(GroES)2 complex is not formed. However, this conventional view has been challenged by the recent reports indicating that such symmetric complexes can be formed in the GroEL-GroES reaction cycle. In this review, we discuss the studies of the symmetric GroEL-(GroES)2 complex, focusing on the molecular mechanism underlying its formation. We also suggest that GroEL can be involved in two types of reaction cycles (asymmetric or symmetric) and the type of cycle used depends on the concentration of non-native substrate proteins.

| S-EPMC5042173 | biostudies-literature

Molecular phylogenetic analysis of Orientia tsutsugamushi based on the groES and groEL genes.

Project description:DNA sequences encoding the GroES and GroEL proteins of Orientia tsutsugamushi were amplified by the PCR and sequenced. Pairwise alignment of full-length groES and groEL gene sequences indicated high sequence similarity (90.4-100% and 90.3-100%) in O. tsutsugamushi, suggesting that these genes are good candidates for the molecular diagnosis and phylogenetic analysis of scrub typhus. Comparisons of the 56-kD type-specific antigen (TSA) protein gene and the groES and groEL genes showed that genotypes based on the 56-kD TSA gene were not related to a cluster containing the groES and groEL genes in a dendrogram, suggesting that a gene rearrangement may be associated with homologous recombination in mites.

| S-EPMC3822374 | biostudies-literature

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