Development of free-energy-based models for chaperonin containing TCP-1 mediated folding of actin

Abstract

A free-energy-based approach is used to describe the mechanism through which chaperonincontaining TCP-1 (CCT) folds the filament-forming cytoskeletal protein actin, which is one of its primary substrates. The experimental observations on the actin folding and unfolding pathways are collated and then re-examined from this perspective, allowing us to determine the position of the CCT intervention on the actin free-energy folding landscape. The essential role for CCT in actin folding is to provide a free-energy contribution from its ATP cycle, which drives actin to fold from a stable, trapped intermediate I 3 , to a less stable but now productive folding intermediate I 2 . We develop two hypothetical mechanisms for actin folding founded upon concepts established for the bacterial type I chaperonin GroEL and extend them to the much more complex CCT system of eukaryotes. A new model is presented in which CCT facilitates free-energy transfer through direct coupling of the nucleotide hydrolysis cycle to the phases of actin substrate maturation.

Interactions between the actin filament capping and severing protein gelsolin and the molecular chaperone CCT: evidence for nonclassical substrate interactions

“…However, it is probable that the majority of CCT oligomers will be involved in folding the highly abundant substrate proteins actin and tubulin. In the case of actin, it appears that CCT is required to overcome a particular kinetic barrier in the later stages of its folding pathway (Altschuler and Willison 2008). This is consistent with the observation that interactions between CCT and actin are charged/polar in nature (Hynes and Willison 2000) rather than binding, being mediated via nonspecific hydrophobic sites.…”

Yeast Phosducin-Like Protein 2 Acts as a Stimulatory Co-Factor for the Folding of Actin by the Chaperonin CCT via a Ternary Complex

“…2d), which is expected since I 3 is the only form of actin in Scheme 1 that binds CCT and the rate of unassisted folding from I 3 to I 2 is immeasurably slow. 3,22 Sedimentation analysis of 1 μM ACT1 EDTA unfolded in the presence of 370 nM CCT shows that CCT is efficiently loaded with actin in this manner (Fig. 2e).…”

“…A slow cycle may increase the yield of actin by allowing its kinetic and thermodynamic folding and unfolding barriers to be breached by the CCT free-energy cycle. 3 A biological consequence of the intricate nature of CCT allostery may be that this slow cycle affords opportunity for further allosteric regulation of folding activity by protein-binding partners such as Plp2p and small molecules. Using the CCT-ACT1-PLP2 assay, we are establishing a screen of key metabolites of yeast 40 to discover if any are allosteric regulators of CCT.…”

“…[1][2][3] The eukaryotic cytosolic chaperonin-containing TCP-1 (CCT; also known as TRiC) is known to fold actins and tubulins and a small number of other proteins, including members of the WD40-propeller fold family involved in cell cycle control. 4 In the yeast Saccharomyces cerevisiae, the interaction network of CCT presently comprises 136 genes/proteins, 5 and we are in the process of classifying which members are bona fide substrates, co-factors or ancillary partners.…”

Reconciling theories of chaperonin accelerated folding with experimental evidence

“…After submission, we were informed of new experimental evidence that a number of these proteins may not be obligate GroEL substrates in GroEL/GroES-depleted cells. Active cage Chaperonins may accelerate folding by modifying the static environment in which proteins fold by, for example, smoothing the energy landscape [16,115,116], reducing the entropy of the unfolded state, [19,20,103,[116][117][118][119][120][121][122][123][124], providing new pathways for folding [11,19,20,103,110,124,125], or modulating the solvent behavior [122,[126][127][128][129][130][131].…”