Chaperonin complex with a newly folded protein encapsulated in the folding chamber

Abstract

A subset of essential cellular proteins requires the assistance of chaperonins (in Escherichia coli, GroEL and GroES), double-ring complexes in which the two rings act alternately to bind, encapsulate and fold a wide range of nascent or stress-denatured proteins. This process starts by the trapping of a substrate protein on hydrophobic surfaces in the central cavity of a GroEL ring. Then, binding of ATP and co-chaperonin GroES to that ring ejects the non-native protein from its binding sites, through forced unfolding or other major conformational changes, and encloses it in a hydrophilic chamber for folding. ATP hydrolysis and subsequent ATP binding to the opposite ring trigger dissociation of the chamber and release of the substrate protein. The bacteriophage T4 requires its own version of GroES, gp31, which forms a taller folding chamber, to fold the major viral capsid protein gp23 (refs 16-20). Polypeptides are known to fold inside the chaperonin complex, but the conformation of an encapsulated protein has not previously been visualized. Here we present structures of gp23-chaperonin complexes, showing both the initial captured state and the final, close-to-native state with gp23 encapsulated in the folding chamber. Although the chamber is expanded, it is still barely large enough to contain the elongated gp23 monomer, explaining why the GroEL-GroES complex is not able to fold gp23 and showing how the chaperonin structure distorts to enclose a large, physiological substrate protein.

Figure 1. Asymmetric reconstructions of GroEL with non-native gp23 in one or both rings

a-d: Binary complex with gp23 in one ring (red density) with the crystal structures of GroEL domains fitted into the maps, shown from the side (a), as a central section (b), from the top (c) and from the bottom (d). e-h: The same views of the binary complex with gp23 in both rings (red density). The EM density maps were sharpened between 20 and 10 Å. Automated docking of the atomic coordinates of the 42 GroEL domains as rigid bodies into each complex gave excellent fits to the maps, with hinge residues of neighbouring domains in proximity to each other, except for a few regions such as the intermediate domains of some subunits. Helices H and I are shown as cyan cylinders. The C-termini of some subunits are visible and either contact the substrate (b, upper ring) or bend away from it (f, lower ring). Interaction of the flexible GroEL C-termini with substrates is consistent with earlier reports,. The resolution of the maps is around 11 Å at 0.5 Fourier shell correlation.

Figure 2. Asymmetric reconstructions of GroEL-gp31 without visible substrate, with gp23 in the open ring and with gp23 in both rings

a-c: Empty ternary complex with the crystal structures of GroEL and gp31 fitted as 49 individual domains shown from the side (a), as a central section (b) and from the bottom (c). d-f: Same views of the trans-bound ternary complex. g-i: Same views of the cis/trans bound ternary complex. Gp23 density in the trans rings is shown in red and in the cis ring in green. The resolution of the maps is around 10 Å at 0.5 Fourier shell correlation and they were therefore sharpened between 20 and 10 Å. The GroEL and gp31 domain coordinates fit very well into the density maps, with only a few minor mismatches and no significant perturbation from 7-fold symmetry.

Figure 3. Folding chambers of the GroEL-gp23-gp31 complexes

a-c, side view sections and d-f, cross sections through the apical domains of the folding chambers (at the position of the red dotted lines in a-c). Empty complex (a and d), the trans-occupied complex (b and e) and the cis/trans occupied complex (c and f). The red arrows in a-c show the loss of density at the contact between gp31 and GroEL. Correspondingly, the dotted red circles in d-f are all the same size (45 Å in diameter) and highlight the expansion of the apical domain ring in the cis/trans complex and the contraction of the trans-occupied apical domain ring. The major domain of the gp24 structure fits very well into the cis substrate density (c, f), but not the mobile insertion domain of gp24 in the extended conformation seen in the crystal structure, where it makes an inter-subunit contact. In this position it clashes with the GroEL C-termini, but there is clearly space available for it closer to the major domain.

Figure 4. Substrate densities isolated from the binary and ternary complexes

Top and side views of substrate densities isolated from the GroEL-gp23 binary complex (a), the GroEL-gp23₂ binary complex (b), the trans-only GroEL-gp23-gp31 ternary complex (c) and the cis ternary complex (d) compared to the low resolution filtered density of the gp24 crystal structure (e). The isolated substrate densities were low-pass filtered at 15 Å and their approximate molecular masses were determined at a density threshold of 1σ of the complete complex. A larger observed mass in the class average reflects a more homogeneous class and therefore a more consistent structure for that sub-population. The green line in (e) indicates the major domain of gp24.