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Topologies of a substrate protein bound to the chaperonin GroEL.

ABSTRACT: The chaperonin GroEL assists polypeptide folding through sequential steps of binding nonnative protein in the central cavity of an open ring, via hydrophobic surfaces of its apical domains, followed by encapsulation in a hydrophilic cavity. To examine the binding state, we have classified a large data set of GroEL binary complexes with nonnative malate dehydrogenase (MDH), imaged by cryo-electron microscopy, to sort them into homogeneous subsets. The resulting electron density maps show MDH associated in several characteristic binding topologies either deep inside the cavity or at its inlet, contacting three to four consecutive GroEL apical domains. Consistent with visualization of bound polypeptide distributed over many parts of the central cavity, disulfide crosslinking could be carried out between a cysteine in a bound substrate protein and cysteines substituted anywhere inside GroEL. Finally, substrate binding induced adjustments in GroEL itself, observed mainly as clustering together of apical domains around sites of substrate binding.

SUBMITTER: Elad N

PROVIDER: S-EPMC1885994 | biostudies-literature | 2007 May

REPOSITORIES: biostudies-literature

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Topologies of a substrate protein bound to the chaperonin GroEL.

Elad Nadav N Farr George W GW Clare Daniel K DK Orlova Elena V EV Horwich Arthur L AL Saibil Helen R HR

Molecular cell 20070501 3

The chaperonin GroEL assists polypeptide folding through sequential steps of binding nonnative protein in the central cavity of an open ring, via hydrophobic surfaces of its apical domains, followed by encapsulation in a hydrophilic cavity. To examine the binding state, we have classified a large data set of GroEL binary complexes with nonnative malate dehydrogenase (MDH), imaged by cryo-electron microscopy, to sort them into homogeneous subsets. The resulting electron density maps show MDH asso ...[more]

PMID: 17499047

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Project description:The double ring-shaped chaperonin GroEL binds a wide range of non-native polypeptides within its central cavity and, together with its cofactor GroES, assists their folding in an ATP-dependent manner. The conformational cycle of GroEL/ES has been studied extensively but little is known about how the environment in the central cavity affects substrate conformation. Here, we use the von Hippel-Lindau tumor suppressor protein VHL as a model substrate for studying the action of the GroEL/ES system on a bound polypeptide. Fluorescent labeling of pairs of sites on VHL for fluorescence (Förster) resonant energy transfer (FRET) allows VHL to be used to explore how GroEL binding and GroEL/ES/nucleotide binding affect the substrate conformation. On average, upon binding to GroEL, all pairs of labeling sites experience compaction relative to the unfolded protein while single-molecule FRET distributions show significant heterogeneity. Upon addition of GroES and ATP to close the GroEL cavity, on average further FRET increases occur between the two hydrophobic regions of VHL, accompanied by FRET decreases between the N- and C-termini. This suggests that ATP- and GroES-induced confinement within the GroEL cavity remodels bound polypeptides by causing expansion (or racking) of some regions and compaction of others, most notably, the hydrophobic core. However, single-molecule observations of the specific FRET changes for individual proteins at the moment of ATP/GroES addition reveal that a large fraction of the population shows the opposite behavior; that is, FRET decreases between the hydrophobic regions and FRET increases for the N- and C-termini. Our time-resolved single-molecule analysis reveals the underlying heterogeneity of the action of GroES/EL on a bound polypeptide substrate, which might arise from the random nature of the specific binding to the various identical subunits of GroEL, and might help explain why multiple rounds of binding and hydrolysis are required for some chaperonin substrates.

Dataset Information

Topologies of a substrate protein bound to the chaperonin GroEL.

Publications

Topologies of a substrate protein bound to the chaperonin GroEL.

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