A two-domain folding intermediate of RuBisCO in complex with the GroEL chaperonin

The chaperonins (GroEL and GroES in Escherichia coli) are ubiquitous molecular chaperones that assist a subset of essential substrate proteins to undergo productive folding to the native state. Using single particle cryo EM and image processing we have examined complexes of E. coli GroEL with the stringently GroE-dependent substrate enzyme RuBisCO from Rhodospirillum rubrum. Here we present snapshots of non-native RuBisCO - GroEL complexes. We observe two distinct substrate densities in the binary complex reminiscent of the two-domain structure of the RuBisCO subunit, so that this may represent a captured form of an early folding intermediate. The occupancy of the complex is consistent with the negative cooperativity of GroEL with respect to substrate binding, in accordance with earlier mass spectroscopy studies. [Abstract copyright: Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Structure and allostery of the Chaperonin GroEL

Chaperonins are intricate allosteric machines formed of two back-to-back, stacked rings of subunits presenting end cavities lined with hydrophobic binding sites for nonnative polypeptides. Once bound, substrates are subjected to forceful, concerted movements that result in their ejection from the binding surface and simultaneous encapsulation inside a hydrophilic chamber that favors their folding. Here, we review the allosteric machine movements that are choreographed by ATP binding, which triggers concerted tilting and twisting of subunit domains. These movements distort the ring of hydrophobic binding sites and split it apart, potentially unfolding the multiply bound substrate. Then, GroES binding is accompanied by a 100° twist of the binding domains that removes the hydrophobic sites from the cavity lining and forms the folding chamber. ATP hydrolysis is not needed for a single round of binding and encapsulation but is necessary to allow the next round of ATP binding in the opposite ring. It is this remote ATP binding that triggers dismantling of the folding chamber and release of the encapsulated substrate, whether folded or not. The basis for these ordered actions is an elegant system of nested cooperativity of the ATPase machinery. ATP binds to a ring with positive cooperativity, and movements of the interlinked subunit domains are concerted. In contrast, there is negative cooperativity between the rings, so that they act in alternation. It is remarkable that a process as specific as protein folding can be guided by the chaperonin machine in a way largely independent of substrate protein structure or sequence

Chaperones and protein folding

The folding and translocation of many newly synthesized proteins in the cell is kinetically assisted by ubiquitous, abundant, specialized proteins known as molecular chaperones. These components generally recognize hydrophobic surfaces, exposed specifically by non-native conformations, through their own solvent-exposed hydrophobic surfaces, with different classes of chaperone recognizing such surfaces in the context of extended (Hsp70) vs. collapsed (Hsp60/chaperonin) topology of substrate protein. Such binding prevents substrate proteins from misfolding and from forming multimolecular aggregates. Chaperone-bound proteins are then released from Hsp70 and Hsp60 machines via the binding of ATP to chaperone domains physically separated from the substrate protein binding domains, via allosterically directed conformational changes. Molecular chaperones also act under stress conditions, where polypeptide chains are subject to misfolding, preventing aggregation and restoring the native state. The small heat shock proteins (sHsps) are oligomeric assemblies that participate with the other chaperones in binding non-native states under such conditions. Finally, Hsp90 is an abundant clamp-shaped chaperone that participates in binding and maturation of a variety of substrate proteins via an ATP-directed cycle. This chapter reviews the structure and mechanism of action of these chaperones, with special attention directed to the variety of biophysical methods employed to reaching our current understanding