Dataset Information

Cryo-EM structures of GroEL:ES₂ with RuBisCO visualize molecular contacts of encapsulated substrates in a double-cage chaperonin.

ABSTRACT: The GroEL/GroES chaperonin system assists the folding of many proteins, through conformational transitions driven by ATP hydrolysis. Although structural information about bullet-shaped GroEL:ES₁ complexes has been extensively reported, the substrate interactions of another functional complex, the football-shaped GroEL:ES₂, remain elusive. Here, we report single-particle cryo-EM structures of reconstituted wild-type GroEL:ES₂ complexes with a chemically denatured substrate, ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO). Our structures demonstrate that native-like folded RuBisCO density is captured at the lower part of the GroEL chamber and that GroEL's bulky hydrophobic residues Phe281, Tyr360, and Phe44 contribute to direct contact with RuBisCO density. In addition, our analysis found that GroEL:ES₂ can be occupied by two substrates simultaneously, one in each chamber. Together, these observations provide insights to the football-shaped GroEL:ES₂ complex as a functional state to assist the substrate folding with visualization.

SUBMITTER: Kim H

PROVIDER: S-EPMC8749442 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Cryo-EM structures of GroEL:ES2 with RuBisCO visualize molecular contacts of encapsulated substrates in a double-cage chaperonin.

Kim Hyunmin H Park Junsun J Lim Seyeon S Jun Sung-Hoon SH Jung Mingyu M Roh Soung-Hun SH

iScience 20211227 1

The GroEL/GroES chaperonin system assists the folding of many proteins, through conformational transitions driven by ATP hydrolysis. Although structural information about bullet-shaped GroEL:ES1 complexes has been extensively reported, the substrate interactions of another functional complex, the football-shaped GroEL:ES2, remain elusive. Here, we report single-particle cryo-EM structures of reconstituted wild-type GroEL:ES2 complexes with a chemically denatured ...[more]

PMID: 35036883

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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

Cryo-EM structures of GroEL:ES₂ with RuBisCO visualize molecular contacts of encapsulated substrates in a double-cage chaperonin.

Publications

Cryo-EM structures of GroEL:ES<sub>2</sub> with RuBisCO visualize molecular contacts of encapsulated substrates in a double-cage chaperonin.

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