GroEL and CCT are catalytic unfoldases mediating out-of-cage polypeptide refolding without ATP

Abstract

Chaperonins are cage-like complexes in which nonnative polypeptides prone to aggregation are thought to reach their native state optimally. However, they also may use ATP to unfold stably bound misfolded polypeptides and mediate the out-of-cage native refolding of large proteins. Here, we show that even without ATP and GroES, both GroEL and the eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) can unfold stable misfolded polypeptide conformers and readily release them from the access ways to the cage. Reconciling earlier disparate experimental observations to ours, we present a comprehensive model whereby following unfolding on the upper cavity, in-cage confinement is not needed for the released intermediates to slowly reach their native state in solution. As oversticky intermediates occasionally stall the catalytic unfoldase sites, GroES mobile loops and ATP are necessary to dissociate the inhibitory species and regenerate the unfolding activity. Thus, chaperonin rings are not obligate confining antiaggregation cages. They are polypeptide unfoldases that can iteratively convert stable off-pathway conformers into functional proteins.

Intrinsic unfoldase/foldase activity of the chaperonin GroEL directly demonstrated using multinuclear relaxation-based NMR

“…3. The range of parameters reported for [ 15 N/ 1 H N /AILV- 13 CH 3 ]/deuterated-SH3 Mut covers the variation observed between ( 15 N, 1 H N ) and 13 C methyl probes (details of both sets of fits in SI Theory). Note that the values of the rate constants have been appropriately rounded and hence do not exactly satisfy the principle of detailed balance (compare SI Theory, Eq.…”

“…SH3 samples (both wild type and triple mutant) were isotopically labeled either with 15 N or with 15 N, 2 H (except 1 H N protons, which are protonated through exchange with water) and selective 13 C methyl labeling of Ala-β, Ile-δ1, Leu-δ, and Val-γ methyl groups (full details in SI Materials and Methods). Purification and expression of the SH3 domains, GroEL and Rubisco were carried out as described in SI Materials and Methods.…”

“…To gain further insight into the catalytic activity of apo GroEL and to probe the location of the SH3 Mut binding site, we included additional probes ( 1 H N and 13 C methyl nuclei) in the analysis and recorded 1 H N , 15 N, and 13 (Fig. S5), as well as complementary 15 N/ 13 C methyl ΔR 2 ( Fig.…”

“…suggested that GroEL may possess intrinsic unfoldase activity but the conclusions were drawn from indirect evidence and/or involved poorly defined, heterogeneous systems (12,13). The intrinsic unfoldase activity of apo GroEL presented here is distinct from the encapsulation cycle and supports the proposed iterative annealing mechanism of GroEL activity (24,25), whereby an incorrectly folded, kinetically trapped substrate is unfolded on the internal surface of GroEL and subsequently released either into the GroEL cavity or into free solution where further attempts at correct folding can occur.…”

“…Further, encapsulation does not appear to be an absolute requirement for successful re/folding (8). Moreover, hydrogen/deuterium exchange experiments on several protein substrates (9)(10)(11)(12) and fluorescence-based refolding experiments (13) suggest that the prototypical chaperonin GroEL may possess intrinsic unfoldase activity. Here we take advantage of a monomeric, nonaggregating, well-defined system-a triple mutant of the Fyn SH3 domain that exists in dynamic equilibrium between the major native state and a sparsely populated folding intermediate (14,15)-to directly demonstrate, using NMR relaxation-based methods (16), the ability of apo GroEL to accelerate the interconversion between these two states by almost three orders of magnitude.…”