Role of the Helical Protrusion in the Conformational Change and Molecular Chaperone Activity of the Archaeal Group II Chaperonin

Abstract

To elucidate the exact role of the helical protrusion of a group II chaperonin in its molecular chaperone function, three deletion mutants of the chaperonin from a hyperthermophilic archaeum (Thermococcus sp. strain KS-1) lacking one-third, two-thirds, and the whole of the helical protrusion were constructed. The helical protrusion is thought to be substituted for the co-chaperonin GroES of a group I chaperonin and to be important for binding to unfolded proteins. Protease sensitivity assays and small angle x-ray scattering experiments were performed to demonstrate the conformation change of the wild type protein and the deletion mutants by adenine nucleotides. Whereas the binding of ATP to the wild type protein induced a structural transition corresponding to the closure of the built-in lid, it did not cause significant structural changes in deletion mutants. Although the mutants effectively protected proteins from thermal aggregation, ATP-dependent protein folding ability was remarkably diminished. We conclude that the helical protrusion is not necessarily important for binding to unfolded proteins, but its ATP-dependent conformational change mediates folding of captured unfolded proteins.Protein folding is assisted by a number of molecular chaperones in vivo. The chaperonins, a ubiquitous class of molecular chaperones, form double ring complexes that mediate the folding of nascent and denatured proteins in an ATP-dependent manner (1, 2). There are two distinct groups of chaperonins: (i) group I chaperonins of eubacteria and endosymbiotic organelles (mitochondria and chloroplasts); and (ii) group II chaperonins of archaea (known as thermosome) and the eukaryotic cytoplasm (known as CCT 1 or TRiC) (3, 4). Despite the relatively low sequence homology between the two groups, their domain architectures are very similar. Both of them are folded into three domains. The equatorial domain contains the ATP binding site and is involved in intra-and inter-ring contacts. The apical domain is involved in the binding to substrate proteins. The intermediate domain connects the equatorial and apical domains of each subunit and transfers the ATP-induced conformational changes from the equatorial to the apical domain (5, 6).The most striking structural difference between them is the lid of the central cavity of the chaperonin complex. The co-chaperonin GroES/cpn-10 serves as a lid for group I chaperonins in a heptameric dome-like structure. GroES/cpn-10 interacts with one or both GroEL rings in an ATP-regulated fashion, thereby sealing the cavity from the outside. It is demonstrated that a polypeptide of up to ϳ57 kDa is able to fold within the GroESsealed cavity (7). On the other hand, there is no GroES-like co-chaperonin for group II chaperonins. Instead, group II chaperonins have a built-in lid, which is composed of an extension of the apical domain called the helical protrusion. The helical protrusion is thought to play the equivalent role of GroES, sealing off the central cavity of the chaperonin complex (6, 8 -12). Furthe...

Sequential Action of ATP-dependent Subunit Conformational Change and Interaction between Helical Protrusions in the Closure of the Built-in Lid of Group II Chaperonins

“…The homo-oligomer of D64A/D393A, CpnD64A/ D393A, showed only trace ATP hydrolysis activity and lacked protein folding ability. The homo-oligomer of ⌬245-276, Cpn⌬helical, did not show any ATP-dependent conformational change and was also deficient in protein folding activity in spite of the fact that it had ATPase activity and was capable of capturing an unfolded protein (17).…”

“…The helical protrusion is not required for substrate binding but is indispensable for ATP-dependent conformational transitions and protein folding (16,17). Similar to the group I chaperonin (16, 18 -20), both archaeal and eukaryotic group II chaperonins demonstrate positive and negative cooperative action with ATP within a ring and between the two rings, respectively (16,18,21,22).…”

Multiple chaperonins in bacteria—novel functions and non-canonical behaviors

“…Although the cellular roles played by the two groups of chaperonins are similar, differences in the tertiary and quaternary structures of these groups of chaperonins suggest distinct mechanisms of encapsulation and action. While the group II chaperonins are assisted by prefoldin (Iizuka et al 2004) and Hsp70 homologues (Cuellar et al 2008) for substrate capture, group I acts independently. In addition, inter-ring allosteric movements also differ between the two groups; while the group I chaperonins show sequential movement, the group II chaperonins exhibit concerted movement.…”

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

“…Alternatively, it has been suggested that the helical protrusions might contain substrate-binding sites, based on their structural flexibility and partial hydrophobic character [6,27]. However, substrate binding is not impaired in a group II chaperonin in which the helical protrusion is deleted [28], which indicates that this region is not essential for binding. A third hypothesis [29], based on evolutionary analyses, proposes that the binding sites reside in the inner face of the closed cavity and consist mostly of charged and polar amino acids.…”