Abstract
Class II chaperonins are essential multisubunit complexes that aid the folding of nonnative proteins in the cytosol of archaea and eukarya. They use energy derived from ATP to drive a series of structural rearrangements that enable polypeptides to fold within their central cavity. These events are regulated by an elaborate allosteric mechanism in need of elucidation. We employed mutagenesis and experimental analysis in concert with in silico molecular dynamics simulations and interface-binding energy calculations to investigate the class II chaperonin from Thermoplasma acidophilum. Here we describe the effects on the asymmetric allosteric mechanism and on hetero-oligomeric complex formation in a panel of mutants in the ATP-binding pocket of the α and β subunits. Our observations reveal a potential model for a nonconcerted folding mechanism optimized for protecting and refolding a range of nonnative substrates under different environmental conditions, starting to unravel the role of subunit heterogeneity in this folding machine and establishing important links with the behavior of the most complex eukaryotic chaperonins.—Shoemark, D. K., Sessions, R. B., Brancaccio, A., Bigotti, M. G. Intraring allostery controls the function and assembly of a hetero-oligomeric class II chaperonin.
Highlights
Chaperonins are large (≈106 Da) oligomeric complexes aiding the folding of non-native polypeptides that are either newly synthesized or misfolded as a consequence of physiological or pathological cellular processes
Based on the nucleotide-bound structure determined at high resolution by X-ray crystallography [4] and/or on analogies with functional mutants in GroEL [35] and yeast CCT [14], two kinds of mutations were inserted in either of the subunits in turn, i.e. those abolishing ATP hydrolysis and those abolishing ATP binding altogether (Fig. 1A and 1B)
We suggest that the amount of sampling required by molecular dynamics to attain an appropriate signal to noise for the energy profiles is beyond current resources
Summary
Chaperonins are large (≈106 Da) oligomeric complexes aiding the folding of non-native polypeptides that are either newly synthesized or misfolded as a consequence of physiological or pathological cellular processes. Chaperonins are composed of two multi-subunit rings stacked back-to-back to form a toroidal cylinder enclosing an internal space, often referred to as the folding chamber This architecture is common to chaperonins from eubacteria and cell organelles (class I) and those from archaea and the eukaryotic cytosol (class II). Despite some degree of sequence variability, the high-resolution crystal and cryo-EM structures of the archaeal [4,5,6] and eukaryotic [7,8,9] class II chaperonins reveal that all subunits share an identical domain organization This consists of an equatorial domain that forms the inter-ring interface and contains the ATP-binding site, an apical domain responsible for the interaction with non-native substrates and containing most of the intra-ring contacts between subunits, and an intermediate domain, connecting the other two, which acts as a hinge that is fundamental for the propagation of allosteric signals. Furthemore, a helical module at the tip of the apical domain acts as a lid closing the folding chamber upon ATP hydrolysis
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