A structural model for GroEL–polypeptide recognition

Buckle, Ashley M.; Zahn, Ralph; Fersht, Alan R.

doi:10.1073/pnas.94.8.3571

Cited by 196 publications

(193 citation statements)

References 35 publications

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“…Thus, after a 2-h incubation, the yields in the case of the wild type and the D3G mutant were 25 and 30%, respectively. The latter result can be explained by a "minichaperone effect" (35,36) due to the instability of the oligomeric structures of the D3G mutant and, although to a lesser extent, of the wild-type mHsp60. In other words, the chaperonins are able to bind unfolded proteins, but upon their dis-oligomerization, the unfolded substrate is released into solution in a form that is able, in vitro, to refold spontaneously.…”

Section: Resultsmentioning

confidence: 99%

The MitCHAP-60 Disease Is Due to Entropic Destabilization of the Human Mitochondrial Hsp60 Oligomer

Parnas

Nadler

Nisemblat

et al. 2009

Journal of Biological Chemistry

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The 60-kDa heat shock protein (mHsp60) is a vital cellular complex that mediates the folding of many of the mitochondrial proteins. Its function is executed in cooperation with the cochaperonin, mHsp10, and requires ATP. Recently, the discovery of a new mHsp60-associated neurodegenerative disorder, Mit-CHAP-60 disease, has been reported. The disease is caused by a point mutation at position 3 (D3G) of the mature mitochondrial Hsp60 protein, which renders it unable to complement the deletion of the homologous bacterial protein in Escherichia coli (Magen, D., Georgopoulos, C., Bross, P., Ang, D., Segev, Y., Goldsher, D., Nemirovski, A., Shahar, E., Ravid, S., Luder, A., Heno, B., Gershoni-Baruch, R., Skorecki, K., and Mandel, H. (2008) Am. J. Hum. Genet. 83, 30 -42). The molecular basis of the MitCHAP-60 disease is still unknown. In this study, we present an in vitro structural and functional analysis of the purified wild-type human mHsp60 and the MitCHAP-60 mutant. We show that the D3G mutation leads to destabilization of the mHsp60 oligomer and causes its disassembly at low protein concentrations. We also show that the mutant protein has impaired protein folding and ATPase activities. An additional mutant that lacks the first three amino acids (N-del), including Asp-3, is similarly impaired in refolding activity. Surprisingly, however, this mutant exhibits profound stabilization of its oligomeric structure. These results suggest that the D3G mutation leads to entropic destabilization of the mHsp60 oligomer, which severely impairs its chaperone function, thereby causing the disease.Type I chaperonins are essential molecular chaperones of the Hsp60 family found in eubacteria, mitochondria, and chloroplasts (1). They are key players in mediating the correct folding of newly translated, translocated, and stress-denatured proteins. The folding function of chaperonins is executed by the coordinated action of two oligomeric proteins, Hsp60 (also named cpn60 and in bacteria GroEL) and its co-chaperonin Hsp10 (also named cpn10 and in bacteria GroES). The GroEL molecule is composed of 14 subunits that form a barrel-like structure that consists of two back-to-back stacked heptameric rings with a large cavity at each end termed the "Anfinsen cage." GroES is a heptameric ring formed by ϳ10-kDa subunits. The co-chaperonin binds to the chaperonin in the presence of ATP and Mg 2ϩ via a short, unstructured, but highly conserved region known as the mobile loop (2, 3). Due to the stability of the GroEL/ES oligomers and the ease with which they can be purified from bacteria, they have become the primary targets for study in the field of chaperonins. As a result, almost all of our knowledge concerning the structure and mechanism of chaperonins, both Hsp60 and Hsp10, is based largely on data from experiments on these E. coli proteins. The chaperonin reaction cycle starts when a non-folded protein substrate binds to the surface of the cavity of one of the GroEL rings. ATP-dependent GroES binding to that (cis) ring causes a dramatic co...

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Section: Resultsmentioning

confidence: 99%

The MitCHAP-60 Disease Is Due to Entropic Destabilization of the Human Mitochondrial Hsp60 Oligomer

Parnas

Nadler

Nisemblat

et al. 2009

Journal of Biological Chemistry

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“…First, GroEL prevents substrate proteins from aggregating by binding unproductive folding intermediates and forces them to unfold to states more committed towards correct folding (Weissman et al, 1994;Ranson et al, 1995;Zahn et al, 1996;Corrales & Fersht, 1996;Buckle et al, 1997). Second, it has been proposed that the central cavity works as an An®nsen cage in which the substrate protein is actively folded (Weissman et al, 1996;Mayhew et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

Conformational changes in the chaperonin GroEL: new insights into the allosteric mechanism 1 1Edited by A. R. Fersht

Groot

Vriend

Berendsen

1999

Journal of Molecular Biology

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Conformational changes are known to play a crucial role in the function of the bacterial GroE chaperonin system. Here, results are presented from an essential dynamics analysis of known experimental structures and from computer simulations of GroEL using the CONCOORD method. The results indicate a possible direct form of inter-ring communication associated with internal¯uctuations in the nucleotide-binding domains upon nucleotide and GroES binding that are involved in the allosteric mechanism of GroEL. At the level of conformational transitions in entire GroEL rings, nucleotide-induced structural changes were found to be distinct and in principle uncoupled from changes occurring upon GroES binding. However, a coupling is found between nucleotide-induced conformational changes and GroES-mediated transitions, but only in simulations of GroEL double rings, and not in simulations of single rings. This provides another explanation for the fact that GroEL functions a double ring system.

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“…Wellordered β-hairpin and extended conformations have been observed by crystallography (8,9), but these conformations were likely selected preferentially during crystallization. NMR-based transferred nuclear Overhauser enhancement studies have reported both helical and hairpin conformations (10)(11)(12), but interpretation is complicated by extensive spin diffusion (13) within GroEL.…”

mentioning

confidence: 99%

Probing the transient dark state of substrate binding to GroEL by relaxation-based solution NMR

Libich

Fawzi

Ying

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

108

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The mechanism whereby the prototypical chaperonin GroEL performs work on substrate proteins has not yet been fully elucidated, hindered by lack of detailed structural and dynamic information on the bound substrate. Previous investigations have produced conflicting reports on the state of GroEL-bound polypeptides, largely due to the transient and dynamic nature of these complexes. Here, we present a unique approach, based on combined analysis of four complementary relaxation-based NMR experiments, to probe directly the "dark" NMR-invisible state of the model, intrinsically disordered, polypeptide amyloid β (Aβ40) bound to GroEL. The four NMR experiments, lifetime line-broadening, dark-state exchange saturation transfer, relaxation dispersion, and small exchange-induced chemical shifts, are dependent in different ways on the overall exchange rates and populations of the free and bound states of the substrate, as well as on residue-specific dynamics and structure within the bound state as reported by transverse magnetization relaxation rates and backbone chemical shifts, respectively. Global fitting of all the NMR data shows that the complex is transient with a lifetime of <1 ms, that binding involves two predominantly hydrophobic segments corresponding to predicted GroEL consensus binding sequences, and that the structure of the bound polypeptide remains intrinsically and dynamically disordered with minimal changes in secondary structure propensity relative to the free state. Our results establish a unique method to observe NMR-invisible dynamic states of GroEL-bound substrates and to describe at atomic resolution the events between substrate binding and encapsulation that are crucial for understanding the normal and stress-related metabolic function of chaperonins. supramolecular machine | protein-protein interactions | conformational sampling

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A structural model for GroEL–polypeptide recognition

Cited by 196 publications

References 35 publications

The MitCHAP-60 Disease Is Due to Entropic Destabilization of the Human Mitochondrial Hsp60 Oligomer

The MitCHAP-60 Disease Is Due to Entropic Destabilization of the Human Mitochondrial Hsp60 Oligomer

Conformational changes in the chaperonin GroEL: new insights into the allosteric mechanism 1 1Edited by A. R. Fersht

Probing the transient dark state of substrate binding to GroEL by relaxation-based solution NMR

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