Visualizing chaperonin function in situ by cryo-electron tomography

Jonathan Wagner, Alonso I. Carvajal, Andreas Bracher, Florian Beck, William Wan, Stefan Bohn, Roman Körner, Wolfgang Baumeister (), Ruben Fernandez-Busnadiego () and F. Ulrich Hartl ()
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Jonathan Wagner: Max Planck Institute of Biochemistry
Alonso I. Carvajal: Max Planck Institute of Biochemistry
Andreas Bracher: Max Planck Institute of Biochemistry
Florian Beck: Max Planck Institute of Biochemistry
William Wan: Vanderbilt University Center for Structural Biology
Stefan Bohn: Max Planck Institute of Biochemistry
Roman Körner: Max Planck Institute of Biochemistry
Wolfgang Baumeister: Max Planck Institute of Biochemistry
Ruben Fernandez-Busnadiego: University Medical Center Göttingen
F. Ulrich Hartl: Max Planck Institute of Biochemistry

Nature, 2024, vol. 633, issue 8029, 459-464

Abstract: Abstract Chaperonins are large barrel-shaped complexes that mediate ATP-dependent protein folding1–3. The bacterial chaperonin GroEL forms juxtaposed rings that bind unfolded protein and the lid-shaped cofactor GroES at their apertures. In vitro analyses of the chaperonin reaction have shown that substrate protein folds, unimpaired by aggregation, while transiently encapsulated in the GroEL central cavity by GroES4–6. To determine the functional stoichiometry of GroEL, GroES and client protein in situ, here we visualized chaperonin complexes in their natural cellular environment using cryo-electron tomography. We find that, under various growth conditions, around 55–70% of GroEL binds GroES asymmetrically on one ring, with the remainder populating symmetrical complexes. Bound substrate protein is detected on the free ring of the asymmetrical complex, defining the substrate acceptor state. In situ analysis of GroEL–GroES chambers, validated by high-resolution structures obtained in vitro, showed the presence of encapsulated substrate protein in a folded state before release into the cytosol. Based on a comprehensive quantification and conformational analysis of chaperonin complexes, we propose a GroEL–GroES reaction cycle that consists of linked asymmetrical and symmetrical subreactions mediating protein folding. Our findings illuminate the native conformational and functional chaperonin cycle directly within cells.

Date: 2024
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DOI: 10.1038/s41586-024-07843-w

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