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.
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.
Project description:The Escherichia coli chaperonin GroEL is a double-ring chaperone that assists protein folding with the aid of GroES and ATP. Asp-398 in GroEL is known as one of the critical residues on ATP hydrolysis because GroEL(D398A) mutant is deficient in ATP hydrolysis (<2% of the wild type) but not in ATP binding. In the archaeal Group II chaperonin, another aspartate residue, Asp-52 in the corresponding E. coli GroEL, in addition to Asp-398 is also important for ATP hydrolysis. We investigated the role of Asp-52 in GroEL and found that ATPase activity of GroEL(D52A) and GroEL(D52A/D398A) mutants was ∼ 20% and <0.01% of wild-type GroEL, respectively, indicating that Asp-52 in E. coli GroEL is also involved in the ATP hydrolysis. GroEL(D52A/D398A) formed a symmetric football-shaped GroEL-GroES complex in the presence of ATP, again confirming the importance of the symmetric complex during the GroEL ATPase cycle. Notably, the symmetric complex of GroEL(D52A/D398A) was extremely stable, with a half-time of ∼ 150 h (∼ 6 days), providing a good model to characterize the football-shaped complex.
Project description:A key constraint on the growth of most organisms is the slow and inefficient folding of many essential proteins. To deal with this problem, several diverse families of protein folding machines, known collectively as molecular chaperones, developed early in evolutionary history. The functional role and operational steps of these remarkably complex nanomachines remain subjects of active debate. Here we present evidence that, for the GroEL-GroES chaperonin system, the non-native substrate protein enters the folding cycle on the trans ring of the double-ring GroEL-ATP-GroES complex rather than the ADP-bound complex. The properties of this ATP complex are designed to ensure that non-native substrate protein binds first, followed by ATP and finally GroES. This binding order ensures efficient occupancy of the open GroEL ring and allows for disruption of misfolded structures through two phases of multiaxis unfolding. In this model, repeated cycles of partial unfolding, followed by confinement within the GroEL-GroES chamber, provide the most effective overall mechanism for facilitating the folding of the most stringently dependent GroEL substrate proteins.
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.
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.
Project description:Two heptamer rings of chaperonin GroEL undergo opening-closing conformational transition in the reaction cycle with the aid of GroES and ATP. We introduced Cys into the GroEL subunit at Ala-384 and Ser-509, which are very close between adjacent GroEL subunits in the open heptamer ring but far apart in the closed heptamer ring. The open ring-specific inter-subunit cross-linking between these Cys indicated that the number of rings in open conformation in GroEL was two in ATP (GroEL(OO)), one in ADP (GroEL(O)), and none in the absence of nucleotide. ADP showed an inhibitory effect on ATP-induced generation of GroEL(OO). The isolated GroEL(O) and GroEL(OO), which lost any bound nucleotide, could bind GroES to form a bullet-shaped 1:1 GroEL-GroES complex and a football-shaped 1:2 GroEL-GroES complex, respectively, even without the addition of any nucleotide. Substrate protein was unable to form a stable complex with GroEL(OO) and did not stimulate ATPase activity of GroEL. These results favor a model of the GroEL reaction cycle that includes a football complex as a critical intermediate.
Project description:In mediating protein folding, chaperonin GroEL and cochaperonin GroES form an enclosed chamber for substrate proteins in an ATP-dependent manner. The essential role of the double ring assembly of GroEL is demonstrated by the functional deficiency of the single ring GroEL(SR). The GroEL(SR)-GroES is highly stable with minimal ATPase activity. To restore the ATP cycle and the turnover of the folding chamber, we sought to weaken the GroEL(SR)-GroES interaction systematically by concatenating seven copies of groES to generate groES(7). GroES Ile-25, Val-26, and Leu-27, residues on the GroEL-GroES interface, were substituted with Asp on different groES modules of groES(7). GroES(7) variants activate ATP activity of GroEL(SR), but only some restore the substrate folding function of GroEL(SR), indicating a direct role of GroES in facilitating substrate folding through its dynamics with GroEL. Active GroEL(SR)-GroES(7) systems may resemble mammalian mitochondrial chaperonin systems.
Project description:Many proteins cannot fold without the assistance of chaperonin machines like GroEL and GroES. The nature of this assistance, however, remains poorly understood. Here we demonstrate that unfolding of a substrate protein by GroEL enhances protein folding. We first show that capture of a protein on the open ring of a GroEL-ADP-GroES complex, GroEL's physiological acceptor state for non-native proteins in vivo, leaves the substrate protein in an unexpectedly compact state. Subsequent binding of ATP to the same GroEL ring causes rapid, forced unfolding of the substrate protein. Notably, the fraction of the substrate protein that commits to the native state following GroES binding and protein release into the GroEL-GroES cavity is proportional to the extent of substrate-protein unfolding. Forced protein unfolding is thus a central component of the multilayered stimulatory mechanism used by GroEL to drive protein folding.
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.
Project description:The GroEL/GroES reaction cycle involves steps of ATP and polypeptide binding to an open GroEL ring before the GroES encapsulation step that triggers productive folding in a sequestered chamber. The physiological order of addition of ATP and nonnative polypeptide, typically to the open trans ring of an asymmetrical GroEL/GroES/ADP complex, has been unknown, although there have been assumptions that polypeptide binds first, allowing subsequent ATP-mediated movement of the GroEL apical domains to exert an action of forceful unfolding on the nonnative polypeptide. Here, using fluorescence measurements, we show that the physiological order of addition is the opposite, involving rapid binding of ATP, accompanied by nearly as rapid apical domain movements, followed by slower binding of nonnative polypeptide. In order-of-addition experiments, approximately twice as much Rubisco activity was recovered when nonnative substrate protein was added after ATP compared with it being added before ATP, associated with twice as much Rubisco protein recovered with the chaperonin. Furthermore, the rate of Rubisco binding to an ATP-exposed ring was twice that observed in the absence of nucleotide. Finally, when both ATP and Rubisco were added simultaneously to a GroEL ring, simulating the physiological situation, the rate of Rubisco binding corresponded to that observed when ATP had been added first. We conclude that the physiological order, ATP binding before polypeptide, enables more efficient capture of nonnative substrate proteins, and thus allows greater recovery of the native state for any given round of the chaperonin cycle.