Functional Communications between the Apical and Equatorial Domains of GroEL through the Intermediate Domain

Abstract

The Escherichia coli GroEL subunit consists of three domains with distinct functional roles. To understand the role of each of the three domains, the effects of mutating a single residue in each domain (Y203C at the apical, T89W at the equatorial, and C138W at the intermediate domain) were studied in detail, using three different enzymes (enolase, lactate dehydrogenase, and rhodanese) as refolding substrates. By analyzing the effects of each mutation, a transfer of signals was detected between the apical domain and the equatorial domain. A signal initiated by the equatorial domain triggers the release of polypeptide from the apical domain. This trigger was independent of nucleotide hydrolysis, as demonstrated using an ATPase-deficient mutant, and, also, the conditions for successful release of polypeptide could be modified by a mutation in the apical domain, suggesting that the polypeptide release mechanism of GroEL is governed by chaperonin-target affinities. Interestingly, a reciprocal signal from the apical domain was suggested to occur, which triggered nucleotide hydrolysis in the equatorial domain. This signal was disrupted by a mutation in the intermediate domain to create a novel ternary complex in which GroES and refolding protein are simultaneously bound in a stable ternary complex devoid of ATPase activity. These results point to a multitude of signals which govern the overall chaperonin mechanism.

GroEL-Substrate-GroES Ternary Complexes Are an Important Transient Intermediate of the Chaperonin Cycle

“…The state of the nucleotide would be important in determining the relative position of this intermediate in the overall chaperonin mechanism. Our initial assumption regarding this question was that the arrested complex would not undergo additional hydrolysis cycles once formed, in other words, that the arrested complex would be an ATPGroEL-rhodanese-GroES complex (7). Surprisingly however, the results shown in Fig.…”

“…Mutations in each of these domains result in alterations in the facet of the chaperonin mechanism that is most reflective of the specific role of each domain (4, 6 -10). Many subtle changes may be seen when mutations and other alterations are introduced into the intermediate domain, as this has the effect of altering the cooperation of the other two domains to complete a functional chaperonin cycle (7,11).…”

“…In a previous study, our laboratory evaluated the effects of three mutant GroEL proteins whose mutations were each located in a different domain of the GroEL subunit (7). Two of these mutants, GroEL T89W and GroEL Y203C, produced predictable effects in their respective domains: GroEL T89W (12), in the ATP hydrolytic abilities localized in the equatorial domain, and GroEL Y203C, in the substrate-binding characteristics of the apical domain.…”

Stopped-flow Fluorescence Analysis of the Conformational Changes in the GroEL Apical Domain

“…Experimental evidence of chaperonin ternary complexes, where GroES and refolding protein molecules are simultaneously bound to the same ring of GroEL, have been reported. In our own studies, we have succeeded in detecting a productive ternary complex of GroEL, GroES, and refolding protein by utilizing GroEL C138W, a mutant chaperonin capable of temperature-dependent arrest of the chaperonin cycle (19,25). The ability to simultaneously form interactions between GroES and refolding protein in a single ring of GroEL would conceivably ensure the successful encapsulation of the refolding protein within the GroEL central cavity, preventing non-constructive diffusion of the protein molecule into the surrounding medium and thereby increasing folding efficiency.…”

Heat-shock protein 60 is required for blastema formation and maintenance during regeneration

“…The mutant GroEL V300E was constructed by using the QuikChange site-directed mutagenesis kit (Stratagene). WT GroEL, phage growth E small (GroES), and GroEL V300E proteins were purified as described (24). In vitro functional assay were performed as described (24,25).…”