Three-dimensional reconstruction from cryoelectron micrographs of the eukaryotic cytosolic chaperonin CCT complexed to tubulin shows that CCT interacts with tubulin (both the a and b isoforms) using ®ve speci®c CCT subunits. The CCT±tubulin interaction has a different geometry to the CCT±actin interaction, and a mixture of shared and unique CCT subunits is used in binding the two substrates. Docking of the atomic structures of both actin and tubulin to their CCT-bound conformation suggests a common mode of chaperonin±substrate interaction. CCT stabilizes quasi-native structures in both proteins that are open through their domain-connecting hinge regions, suggesting a novel mechanism and function of CCT in assisted protein folding. Keywords: actin/chaperonin/electron microscopy/protein folding/tubulin IntroductionFolding of many proteins in vivo requires interaction with macromolecular complexes known as chaperonins. These proteins are ubiquitous oligomeric assemblies that have been classi®ed into two distinct families that share limited but signi®cant sequence homology: type I, present in eubacteria and endosymbiotic organelles, and of which the bacterial GroEL is the best known representative; and type II, present in archaebacteria and the eukaryotic cytosol, which are represented by the thermosome and CCT (chaperonin containing TCP-1), respectively (Bukau and Horwich, 1998;Gutsche et al., 1999;Willison, 1999). Most of the chaperonins share a common architecture, a cylinder made up of two back-to-back stacked rings, each one enclosing a cavity where folding takes place. The atomic structures of GroEL (Braig et al., 1994) and the type II thermosome (Ditzel et al., 1998) have revealed a common subunit architecture consisting of three domains: apical, intermediate and equatorial. The equatorial domain provides most of the intra-and inter-ring interactions and contains the binding site for ATP, the hydrolysis of which is necessary for the working cycle of the chaperonin, while the apical domain is involved in substrate binding and undergoes large conformational changes during the folding cycle. There are, however, numerous differences between type I and type II chaperonins, one of which is the absence of co-chaperonins for type II family members, whose role in the closure of the cavity during the chaperonin working cycle is ful®lled instead by a helical protrusion in the apical domain (Klumpp et al., 1997;Ditzel et al., 1998;Llorca et al., 1999a). Another important difference is related to the degree of complexity of the chaperonin ring, ranging from the seven identical subunits of type I chaperonin GroEL to eight different polypeptide subunits in the case of the type II chaperonin CCT. The most important difference between these two chaperonins is, however, related to their substrate speci®city: whereas GroEL interacts with a broad range of substrates (Houry et al., 1999) using a non-speci®c recognition mechanism based on hydrophobic interactions (Bukau and Horwich, 1998;Chen and Sigler, 1999;Shtilerman et al., 19...

Folding to completion of actin and tubulin in the eukaryotic cytosol requires their interaction with cytosolic chaperonin CCT [chaperonin containing tailless complex polypeptide 1 (TCP-1)]. Three-dimensional reconstructions of nucleotide-free CCT complexed to either actin or tubulin show that CCT stabilizes both cytoskeletal proteins in open and quasi-folded conformations mediated through interactions that are both subunit speci®c and geometry dependent. Here we ®nd that upon ATP binding, mimicked by the non-hydrolysable analog AMP-PNP (5¢-adenylylimido-diphosphate), to both CCT±a-actin and CCT± b-tubulin complexes, the chaperonin component undergoes concerted movements of the apical domains, resulting in the cavity being closed off by the helical protrusions of the eight apical domains. However, in contrast to the GroE system, generation of this closed state does not induce the release of the substrate into the chaperonin cavity, and both cytoskeletal proteins remain bound to the chaperonin apical domains. Docking of the AMP-PNP±CCT-bound conformations of a-actin and b-tubulin to their respective native atomic structures suggests that both proteins have progressed towards their native states.

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In order to determine the role of the C-terminal helix in the folding and stability of yeast phosphoglycerate kinase, a mutant deleted of the 12 C-terminal residues (PGK delta 404-415) was constructed. This mutant folds in a conformation very similar to that of the wild-type protein, but exhibits a very low activity (0.1% of that of the wild-type enzyme). The main structural effect of the deletion of the C-terminal helix is an increase in flexibility of the whole protein and a decrease in stability by about 5 kcal/mol. The structural properties of the truncated protein are very similar, at least qualitatively, to those in the isolated domains. The accessibility of the thiol group of Cys 97 is identical to that in the isolated N-domain. The large solvent effect on the tryptophan fluorescence in the native protein at very low concentration of denaturant reveals an increase of flexibility of the C-domain, similar to that observed on the isolated C-domain. NMR measurements show that the pH dependence of His C2H and C4H chemical shifts in the truncated protein perfectly matches those of the isolated domains. The addition of the missing peptide provokes a 40-fold increase in enzyme activity at saturation. A dissociation constant of 80 microM was determined. This peptide, which displays a random structure in solution, folds in a helical structure in the region 405-410 as assessed by TRNOESY. All these results show that the C-terminal part of yeast phosphoglycerate kinase is not necessary for most of the initial folding steps but acts to lock the C-domain on the N-domain, thus ensuring the expression of full enzyme activity. Without this sequence, the protein has the sum of the properties of the two isolated domains.

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