The crystal structure of yeast CCT reveals intrinsic asymmetry of eukaryotic cytosolic chaperonins

Abstract

The cytosolic chaperonin CCT is a 1-MDa protein-folding machine essential for eukaryotic life. The CCT interactome shows involvement in folding and assembly of a small range of proteins linked to essential cellular processes such as cytoskeleton assembly and cell-cycle regulation. CCT has a classic chaperonin architecture, with two heterogeneous 8-membered rings stacked back-to-back, enclosing a folding cavity. However, the mechanism by which CCT assists folding is distinct from other chaperonins, with no hydrophobic wall lining a potential Anfinsen cage, and a sequential rather than concerted ATP hydrolysis mechanism. We have solved the crystal structure of yeast CCT in complex with actin at 3.8 Å resolution, revealing the subunit organisation and the location of discrete patches of co-evolving 'signature residues' that mediate specific interactions between CCT and its substrates. The intrinsic asymmetry is revealed by the structural individuality of the CCT subunits, which display unique configurations, substrate binding properties, ATPbinding heterogeneity and subunit-subunit interactions. The location of the evolutionarily conserved N-terminus of Cct5 on the outside of the barrel, confirmed by mutational studies, is unique to eukaryotic cytosolic chaperonins.

Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro

“…4). The morphology of purified human TRiC was consistent with that of purified TRiC reported in the literature (Cong et al 2010;Dekker et al 2011).…”

“…Purification of endogenous TRiC from rabbit reticulocytes has also been effective (Frydman et al 1994;Gao et al 1992;Nimmesgern and Hartl 1993;Norcum 1996). More recently, purification of exogenously tagged yeast TRiC in yeast has been developed (Dekker et al 2011;Pappenberger et al 2006), along with purification of endogenous yeast TRiC by exogenously tagging an interacting protein (Leitner et al 2012). Co-expression of all eight human CCT subunits in baby hamster kidney cells has been attempted but resulted in very low yields (Machida et al 2012).…”

“…Investigations of the arrangement of the CCT subunits in TRiC purified from different species have given conflicting results (Cong et al 2010;Dekker et al 2011). However, a recent novel mass spectrometry method established that bovine TRiC purified from testes tissue and yeast TRiC purified via an interacting protein had the same arrangement with CCT2 and CCT6 forming the homotypic contacts (Leitner et al 2012).…”

Firefly luciferase mutants as sensors of proteome stress

“…It has been shown in vitro that GroEL can bind about half of the total soluble proteins of E. coli in denatured state (Viitanen et al, 1992) and in vivo approximately 10-15% of all cytoplasmic proteins (under normal growth) and upto 30% of stress denatured proteins interact with GroEL (Ewalt et al, 1997;Houry et al, 1999;Kerner et al, 2005). (Dekker et al, 2011).…”

Dynamics, flexibility, and allostery in molecular chaperonins

“…Recent years have witnessed a great deal of structural information derived from X-ray and EM data [10,11,[72][73][74][75][76][77][78] (Fig. 1B and C).…”