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

Abstract

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...

Facilitated Oligomerization of Mycobacterial GroEL: Evidence for Phosphorylation-Mediated Oligomerization

“…Detailed studies have revealed mechanistic and physiological characteristics of the isologous ring form of the GroEL/S machine (11,42,45,53,54). According to the current understanding, the following properties of GroEL are significant in controlling its activity: (i) oligomerization mediated by the equatorial domain, resulting in the formation of the folding chamber and the encapsulation of substrate polypeptides (48, 59); (ii) recognition of substrate polypeptides medi- (32,63,64); and (iv) ATP hydrolysis (19,55). Impairment of any of these properties significantly alters the functioning of GroEL (37,40).…”

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

“…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).…”

Kinetic Model for the Coupling between Allosteric Transitions in GroEL and Substrate Protein Folding and Aggregation

“…To explore the full effect of GroEL dynamics, from now on, we use the coupled time-dependent equations [Eq. (12)]. In vitro experiments can be used to measure the time-dependent yield of the native state of SPs as a function of GroEL, GroES, and ATP concentrations.…”

“…1 that it is important to account for the coupling between the complex allosteric transitions that the GroEL particle undergoes and the fate of the SP during the reaction cycle in order to describe the workings of the GroEL-GroES machine. 4,[12][13][14][15][16] Studies that artificially simplify the processes in the GroEL cycle ( Fig. 1), such as using SPs that do not require GroES or mutants from which GroES does not dissociate on biologically relevant timescales, while insightful, cannot accurately represent the mechanism of rescue of stringent SPs under nonpermissive conditions.…”