Folding with and without encapsulation by cis- and trans-only GroEL-GroES complexes

A conundrum has arisen in the study of the structural states of the GroEL-GroES chaperonin machine: When either ATP or ADP is added along with GroES to GroEL, the same asymmetric complex, with one ring in a GroES-domed state, is observed by either x-ray crystallographic study or cryoelectron microscopy. Yet only ATP͞ GroES can trigger productive folding inside the GroES-encapsulated cis cavity by ejecting bound polypeptide from hydrophobic apical binding sites during attendant rigid body elevation and twisting of these domains. Here, we show that this difference occurs because polypeptide substrate in fact presents a load on the apical domains, and, although ATP can counter this load effectively, ADP cannot. We monitored apical domain movement in real time by fluorescence resonance energy transfer (FRET) between a fixed equatorial fluorophore and one attached to the mobile apical domain. In the absence of bound polypeptide, addition of either ATP͞GroES or ADP͞GroES to GroEL produced the same rapid rate and extent of decrease of FRET (t 1/2 < 1 sec), reflecting similarly rapid apical movement to the same end-state and explaining the results of the structural studies, which were all carried out in the absence of substrate polypeptide. But in the presence of bound malate dehydrogenase or rhodanese, whereas similar rapid and extensive FRET changes were observed with ATP͞GroES, the rate of FRET change with ADP͞GroES was slowed by >100-fold and the extent of change was reduced, indicating that the apical domains opened in a slow and partial fashion. These results indicate that the free energy of ␥-phosphate binding, measured earlier as 43 kcal per mol (1 cal ‫؍‬ 4.184 J) of rings, is required for driving the forceful excursion or ''power stroke'' of the apical domains needed to trigger release of the polypeptide load into the central cavity.

Section: Time-dependent Changes Of Fret: Presence Of Substrate Polypesupporting

confidence: 84%

Substrate polypeptide presents a load on the apical domains of the chaperonin GroEL

Motojima

Chaudhry

Fenton

et al. 2004

“…Our observation that stable misfolded FTrho-like polypeptides, similar to the 82-kDa aconitase (14), need not fully enter the chamber and stay under a sealed GroES lid suggests that large polypeptides with several misfolded domains might be catalytically unfolded domain by domain, as shown to be the case with chimerical rhodanese fused to GFP or dihydrofolate reductase (31). CCT also was suggested to assist multidomain protein refolding in a domain-by-domain manner, thus mimicking optimal cotranslational folding (32).…”

Section: Discussionmentioning

confidence: 54%

“…When FTrho was compelled to confinement in SR1 under a sealed GroES 7 cap, this was counterproductive compared with wild-type GroEL 14 , indicating that for particular substrates, unrestricted out-of-the-cage refolding may be more effective than restricted in-cage refolding. The unfoldase activity was also found to be prone to gradual inhibition by over-sticky intermediates, and the regeneration of the catalytic activity necessitated ATP and the binding of GroES mobile loops.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, ATP is thought to drive the reaction cycle of GroEL and CCT by timely alternating between an in-cage sequestration phase, to promote by confinement the spontaneous refolding of an unfolded polypeptide, and a dissociation phase, to release the natively refolded protein from the cage into the solution. However, exceptions also have been reported of large proteins being released directly from the GroEL cage without encapsulation, upon GroES binding to the opposite ring, possibly leading to out-of-cage refolding (14,15).…”

mentioning

confidence: 99%

See 1 more Smart Citation

GroEL and CCT are catalytic unfoldases mediating out-of-cage polypeptide refolding without ATP

Priya

Sharma

Sood

et al. 2013

Chaperonins are cage-like complexes in which nonnative polypeptides prone to aggregation are thought to reach their native state optimally. However, they also may use ATP to unfold stably bound misfolded polypeptides and mediate the out-of-cage native refolding of large proteins. Here, we show that even without ATP and GroES, both GroEL and the eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) can unfold stable misfolded polypeptide conformers and readily release them from the access ways to the cage. Reconciling earlier disparate experimental observations to ours, we present a comprehensive model whereby following unfolding on the upper cavity, in-cage confinement is not needed for the released intermediates to slowly reach their native state in solution. As oversticky intermediates occasionally stall the catalytic unfoldase sites, GroES mobile loops and ATP are necessary to dissociate the inhibitory species and regenerate the unfolding activity. Thus, chaperonin rings are not obligate confining antiaggregation cages. They are polypeptide unfoldases that can iteratively convert stable off-pathway conformers into functional proteins.

“…This chimera has a molecular mass of Ϸ66 kDa, and it may, therefore, be too large to be encapsulated in the cis cavity underneath GroES (20), although it has been reported that an 86-kDa protein complex can still be encapsulated (21). The chimera's folding can, however, also be assisted by GroES binding to the trans ring (opposite the polypeptide) of GroEL, as shown for other GroES-dependent substrates (22). Moreover, the size of the substrate is not relevant to this study because the effects of the concerted or sequential ATP-induced conformational changes on substrate release were examined in the absence of GroES.…”

Section: Resultsmentioning

confidence: 98%

Concerted ATP-induced allosteric transitions in GroEL facilitate release of protein substrate domains in an all-or-none manner

Kipnis¹,

Papo²,

Haran

et al. 2007

The double-ring chaperonin GroEL mediates protein folding, in conjunction with its helper protein GroES, by undergoing ATPinduced conformational changes that are concerted within each heptameric ring. Here we have examined whether the concerted nature of these transitions is responsible for protein substrate release in an all-or-none manner. Two chimeric substrates were designed, each with two different reporter activities that were recovered after denaturation in GroES-dependent and independent fashions, respectively. The refolding of the chimeras was monitored in the presence of GroEL variants that undergo ATPinduced intraring conformational changes that are either sequential (F44W/D155A) or concerted (F44W). Our results show that release of a protein substrate from GroEL in a domain-by-domain fashion is favored when the intraring allosteric transitions of GroEL are sequential and not concerted.allostery ͉ chaperonins ͉ cooperativity ͉ protein folding T he Escherichia coli chaperonin GroEL is a molecular machine that assists protein folding by undergoing allosteric transitions between protein substrate binding and release states (for reviews, see refs. 1-3). It is made up of two homoheptameric rings, stacked back-to-back, with a cavity at each end (4) in which protein folding can take place in a confining and protective environment. GroEL functions in conjunction with a heptameric ring-shaped cochaperonin, GroES (5), that caps the cavity of the so-called cis ring (6), thereby triggering dissociation of bound protein substrates into the cavity. The allosteric transitions of GroEL are induced by ATP binding that occurs with positive cooperativity within rings and negative cooperativity between rings (7, 8). Experimental data (refs. 9 and 10 and G. Curien, J. Grason, and G. Lorimer, personal communication) and simulations (11) have shown that the intraring allosteric transitions of GroEL are concerted, in accordance with the Monod-WymanChangeux model of cooperativity (12), as proposed in the nested model (7). In contrast, the intraring allosteric transitions of the eukaryotic heterooligomeric chaperonin CCT were recently shown to occur in a sequential fashion (13) in accordance with the Koshland-Némethy-Filmer model of cooperativity (14). The implications of these different allosteric mechanisms for the folding function of chaperonins have not yet been explored.The work described here was undertaken to establish whether the concerted intraring ATP-induced allosteric transitions of GroEL facilitate simultaneous release of different parts of a bound protein substrate, as previously speculated (13). By contrast, it was suggested (13) that ATP-induced sequential conformational changes in CCT may facilitate sequential release and, as a result, domain-by-domain substrate folding. A powerful tool available for investigating this question is a wild-type variant of GroEL that contains a fluorescence reporter introduced by the F44W mutation and has the D155A substitution that converts its ATP-induced intraring allosteric t...