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. 2023 Dec 12;120(50):e2308933120.
doi: 10.1073/pnas.2308933120. Epub 2023 Dec 8.

Structural basis of substrate progression through the bacterial chaperonin cycle

Scott Gardner  1 , Michele C Darrow  2 , Natalya Lukoyanova  1 , Konstantinos Thalassinos  1   3 , Helen R Saibil  1
Affiliations

Structural basis of substrate progression through the bacterial chaperonin cycle

Scott Gardner et al. Proc Natl Acad Sci U S A. .

Abstract

The bacterial chaperonin GroEL-GroES promotes protein folding through ATP-regulated cycles of substrate protein binding, encapsulation, and release. Here, we have used cryoEM to determine structures of GroEL, GroEL-ADP·BeF3, and GroEL-ADP·AlF3-GroES all complexed with the model substrate Rubisco. Our structures provide a series of snapshots that show how the conformation and interactions of non-native Rubisco change as it proceeds through the GroEL-GroES reaction cycle. We observe specific charged and hydrophobic GroEL residues forming strong initial contacts with non-native Rubisco. Binding of ATP or ADP·BeF3 to GroEL-Rubisco results in the formation of an intermediate GroEL complex displaying striking asymmetry in the ATP/ADP·BeF3-bound ring. In this ring, four GroEL subunits bind Rubisco and the other three are in the GroES-accepting conformation, suggesting how GroEL can recruit GroES without releasing bound substrate. Our cryoEM structures of stalled GroEL-ADP·AlF3-Rubisco-GroES complexes show Rubisco folding intermediates interacting with GroEL-GroES via different sets of residues.

Keywords: CryoEM; Rubisco; chaperonins; protein folding.

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Conflict of interest statement

Competing interests statement:M.C.D. was an employee of SPT Labtech, the company that manufactures Chameleon systems.

Figures

Fig. 1.
Fig. 1.
CryoEM structure of GroEL-Rubisco. (A) CryoEM map of GroEL-Rubisco at 4.5 Å. CryoEM density is shown coloured blue (GroEL) and green (Rubisco). (B) Refined atomic model of GroEL and Rubisco density (green) contoured at 8.0σ. The atomic model of GroEL is coloured blue; the substrate-binding helices H and I are coloured red and orange, respectively. (C) CryoEM map of GroEL-Rubisco contoured at a low threshold (5.0σ). The black arrowhead indicates a possible interaction between non-native Rubisco and the GroEL C termini. Percent values in green text represent the Rubisco density compared to that of a folded Rubisco monomer. (D) Contacts between GroEL subunits 1, 2, and 3 (gray density), and non-native Rubisco (green density). Interacting GroEL residues are labelled and shown as stick models.
Fig. 2.
Fig. 2.
CryoEM structure of GroEL-ADP·BeF3-Rubisco. (A) CryoEM map of GroEL-ADP·BeF3-Rubisco at 3.4 Å. The GroEL map (blue) displayed was generated by DeepEMhancer. Density for non-native Rubisco (green) was isolated from the locally filtered map generated by Relion. (B) Refined atomic model of GroEL-ADP·BeF3 and non-native Rubisco density (green). The substrate-binding helices H and I are coloured red and orange, respectively. The asymmetry can be appreciated from the position of helix H in each subunit. (C) Comparison of GroEL-ADP·BeF3 subunit 1 with the published structure of the Rs1 conformation of GroEL-ATP (PDB: 4AAQ). (D) Comparison of GroEL-ADP·BeF3 subunit 2 with the published crystal structure of GroEL-GroES (PDB: 1SVT). (E) Nucleotide binding sites of each GroEL ring, showing ADP·BeF3 in asymmetric ring subunits and ADP in symmetric ring subunits. Overlaid cryoEM density is shown only for the labelled moieties.
Fig. 3.
Fig. 3.
Interactions between GroEL-ADP·BeF3 and non-native Rubisco. (A) Central slices through the GroEL-ADP·BeF3-Rubisco model overlaid with the cryoEM map. GroEL density is coloured transparent gray; Rubisco/GroEL C-terminal density is coloured green. Panels showing lateral slices through the asymmetric ring apical domains (red panel), asymmetric ring equatorial domains (yellow panel), symmetric ring equatorial domains (blue panel), and symmetric ring apical domains (purple panel). (B) Interactions between GroEL apical domains and non-native Rubisco.
Fig. 4.
Fig. 4.
CryoEM structure of GroEL-ADP·AlF3-Rubisco-GroES. (A) CryoEM map of GroEL-ADP·AlF3-Rubisco-GroES at a global resolution of 3.7 Å, filtered by local resolution. (B) Refined atomic model of GroEL-ADP·AlF3-GroES and non-native Rubisco density (green). (C) Molecular contacts between GroEL-GroES and Rubisco. (D) Nucleotide site density in the cis and trans rings.
Fig. 5.
Fig. 5.
Multiple classes of encapsulated Rubisco. Red dashed circles highlight the contact in all four reconstructions between GroEL residue F281 and Rubisco. (A) Reconstruction of class I from 7,202 particles. Panels highlight the K226 and N229 contacts. (B) Reconstruction of class II from 8,237 particles. Panel highlights the F281 contact. (C) Reconstruction of class III from 7,818 particles. Panels highlight the F281, Y360, and E255 contacts. (D) Reconstruction of class IV from 7,708 particles. Panels highlight the E255, F281, and GroES Y71 contacts.
Fig. 6.
Fig. 6.
Modelling Rubisco inside the GroEL-GroES folding chamber. (A) CryoEM map of GroEL-ADP·AlF3-Rubisco-GroES (class II) at a contour level of 7σ. The two domains of the encapsulated Rubisco monomer are coloured purple (NTD) and green (CTD). (B) Comparison between the crystal structure of a Rubisco monomer and the refined model. (C) Refined model of GroEL-ADP·AlF3-Rubisco-GroES overlaid on the class II density at a contour level of 3σ.
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