Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets

Abstract

Chaperonins are key components of the cellular chaperone machinery. These large, cylindrical complexes contain a central cavity that binds to unfolded polypeptides and sequesters them from the cellular environment. Substrate folding then occurs in this central cavity in an ATP-dependent manner. The eukaryotic chaperonin TCP-1 ring complex (TRiC, also called CCT) is indispensable for cell survival because the folding of an essential subset of cytosolic proteins requires TRiC, and this function cannot be substituted by other chaperones. This specificity indicates that TRiC has evolved structural and mechanistic features that distinguish it from other chaperones. Although knowledge of this unique complex is in its infancy, we review recent advances that open the way to understanding the secrets of its folding chamber.

Figure 1

General architecture of group II chaperonins. (a) Ribbon diagram of an α subunit of the thermosome from Thermoplasma acidophilum. The equatorial ATPase domain (red) is linked to the substrate-binding apical domain (yellow) by a flexible hinge or intermediate domain (blue). The helical protrusion, which is unique to group II chaperonins, is in green. (b) Side view of the closed conformation observed in the X-ray structure of the thermosome [10], with subunit domains colored as in (a). Viewed from the side, the oligomeric structure is formed by two octameric rings. In TCP-1 ring complex (TRiC), each ring is composed of eight subunits. (c) Top view of the closed thermosome structure [10] that highlights how the apical protrusions close into an iris-like lid. For clarity, only apical domains are shown, with one domain in red. (d) Bead models of the ATP-induced transition from the open to closed state for group II chaperonins. The model of the nucleotide-free, open state (left) is based on electron tomographic studies on the thermosome. The closed state is from the X-ray structure of the thermosome [10] and presumably reflects the ATP-induced state.

Figure 2

Molecular determinants of TCP-1 ring complex (TRiC)–substrate interaction. (a) Proposed location of substrate-binding sites in the apical domains of TRiC. The individual regions of the three hypothetical substrate-binding sites are highlighted in red. (i) Based on analogy to GroEL. (ii) Based on structural flexibility and hydrophobic character. (iii) Based on amino acid conservation between ortholog but not paralog subunits. (b) Location of TRiC-binding sites in the Von Hippel-Lindau tumor suppressor (VHL) substrate. Folded VHL contains a β domain (cyan) that contains multiple, antiparallel β strands. Boxes 1 and 2 (red), which are required for association with TRiC, are located in adjacent β strands. (c) Location of TRiC-binding sites in the tryptophan-aspartic acid (WD) domain of substrate CDC20. The subset of antiparallel β sheets that is required for association with TRiC is highlighted in red. The WD domain of CDC20 is modeled on its structural homolog β-transducin [63].

Figure 3

Model of the nucleotide cycle of the eukaryotic chaperonin. (a) In the absence of nucleotide, the open complex can bind to unfolded substrates (U-substrate) through binding sites in the central cavity (red lines). (b) ATP binding alone does not produce closure of the lid and significant folding of the substrate, at least in the case of actin. (c) Formation of the trigonal–bipyramidal transition state of the hydrolysis reaction triggers lid closure and confines the substrate in the central cavity. Folding probably occurs at either this stage of the cycle or following scission of the β–γ phosphate bond. (d) Bond scission or inorganic phosphate (P_i) dissociation is likely to trigger reopening of the lid and the release of folded substrate. The cycle is probably asymmetric but the mechanism that keeps both rings in different stages of the hydrolytic cycle is unclear. Consequently, changes in only one of the two rings are shown (blue). Reproduced, with permission, from Ref. [11].