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 E. coli, GroEL and GroES), double-ring complexes in which the two rings act alternately to bind, encapsulate and fold nascent or stress-denatured proteins1,2,3,4,5. This process starts by the trapping of a substrate protein on hydrophobic surfaces in the central cavity of a GroEL ring6,7,8,9,10. 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 folding11,12,13,14. ATP hydrolysis and subsequent ATP binding to the opposite ring trigger dissociation of the chamber and release of the substrate protein15,2. The bacteriophage T4 requires its own version of GroES, gp31, that forms a taller folding chamber, to fold the major viral capsid protein gp2316, 17,18,19,20. 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.Chaperonin-substrate (binary) complexes were formed by rapidly mixing urea-denatured gp23 with GroEL in 2.5-fold molar excess over GroEL oligomer. Ternary complexes were generated by adding gp31 (bacteriophage T4 GroES homologue) and the ATP transition state analogue ADP•AlF 3 . Cryo electron microscopy (EM) data sets of 30-35,000 particles were collected of both preparations and initial 3D maps were obtained by treating each data set as a single structure with 7-fold symmetry. The resulting maps showed GroEL and GroEL-gp31 complexes with some additional densities in the binding cavities (Supplementary figure 1). As in our earlier study on malate dehydrogenase (MDH) folding, we expected the non-native substrate to form heterogeneous and asymmetric complexes with the chaperonins. Therefore we used a combination of multivariate statistical analysis (MSA) The binary complexes were resolved into 5 classes (Supplementary Figure 2 and Supplementary Table 1). In agreement with our mass spectrometry results22, we observed empty GroEL (20% of the images) and GroEL with extra density in either one (40%) or both rings (40%). The classes displaying the largest fraction of the substrate density are shown in Figure 1, with the GroEL subunit domains fitted into the maps as separate rigid bodies. The GroEL rings deviate little from 7-fold symmetry ( Figure 1c, d, g, h and rotational correlation analysis, not shown). This is unlike GroEL-MDH complexes in which the GroEL apical domains are bunched together on the side of the ring w...

Dynamics, flexibility, and allostery in molecular chaperonins

“…4A) [49]. The binding mode is similar for the larger viral capsid protein gp23, which contacts at least five of the seven apical domains within the ring [50]. These studies also show that the sevenfold rotational symmetry within a ring is disrupted when accommodating the substrate protein, and the associated apical motions are suggested to represent the main conformational effects on substrate binding to GroEL [51,52], a similar behavior to that found in CCT [11] (see below).…”

“…Throughout this association with GroES, the polypeptide substrate is released from its hydrophobic binding sites at the apical domains to the newly generated folding chamber, whose volume is now $120,000 Å 3 . In this state, the cavity walls create a highly hydrophobic environment, which effectively minimizes exposure of the substrate protein's hydrophobic residues [50]. The chamber is also proposed to limit the accessible conformational space of the substrate protein in the folding process, and to prevent aggregation and contacts with any other protein in the crowded cell milieu [54].…”

“…Recent EM studies captured the structure of GroEL with bound substrate in open [49,50] and closed [51,52] ring conformations. The EM densities of non-native malate dehydrogenase bound to GroEL (open conformation) suggest several possible binding topologies, ranging from deep inside the cavity to the cavity inlet, where the substrate seems to be restricted to contacting three or four of the seven consecutive GroEL apical domains (Fig.…”

Single-molecule spectroscopy of protein folding in a chaperonin cage

“…1B). Recent cryoelectron microscopy experiments show that substrate proteins bound to GroEL are predominantly localized deep inside the cavity (67,68), a situation that will facilitate interactions with the chaperonin walls in the GroEL-bound state. The particularly strong interactions of rhodanese with GroEL (69,70) are thus likely to increase molecular friction of the substrate protein in the cavity.…”

Folding of large multidomain proteins by partial encapsulation in the chaperonin TRiC/CCT

“…According to cryo-EM and crystal structures, the GroELGroES folding cage can accommodate proteins of up to ∼70-kDa molecular mass (8,9). Indeed, the majority of bona fide GroEL substrates are smaller than 50 kDa (10-12), consistent with an average size of soluble E. coli proteins of ∼35 kDa (Fig.…”