A structural vista of phosducin-like PhLP2A-chaperonin TRiC cooperation during the ATP-driven folding cycle

Abstract

Proper cellular proteostasis, essential for viability, requires a network of chaperones and cochaperones. ATP-dependent chaperonin TRiC/CCT partners with cochaperones prefoldin (PFD) and phosducin-like proteins (PhLPs) to facilitate the folding of essential eukaryotic proteins. Using cryoEM and biochemical analyses, we determine the ATP-driven cycle of TRiC-PFD-PhLP2A interaction. In the open TRiC state, PhLP2A binds to the chamber's equator while its N-terminal H3-domain binds to the apical domains of CCT3/4, thereby displacing PFD from TRiC. ATP-induced TRiC closure rearranges the contacts of PhLP2A domains within the closed chamber. In the presence of substrate, actin and PhLP2A segregate into opposing chambers, each binding to the positively charged inner surfaces formed by CCT1/3/6/8. Notably, actin induces a conformational change in PhLP2A, causing its N-terminal helices to extend across the inter-ring interface to directly contact a hydrophobic groove in actin. Our findings reveal an ATP-driven PhLP2A structural rearrangement cycle within the TRiC chamber to facilitate folding.

The structural basis of eukaryotic chaperonin TRiC/CCT: Action and folding

“…Upon chamber closing, half of the hemisphere of CCT1/3/6/8/ is charged positively, while the other half hemisphere is charged negatively. Figures are adopted from Park et al (2023) . (D) Color-coded diagram of different ATP affinities of individual CCT subunits.…”

“…These PhLP homologs share a common domain structure featuring a central thioredoxin-like domain (TXD) flanked by variable-length, flexible N-terminal domain and C-terminal domain (NTD and CTD, respectively) ( Fig. 4A ) ( Park et al, 2023 ). Each PhLP isoform exhibits unique activity and specificity toward different TRiC substrates.…”

“…Each PhLP isoform exhibits unique activity and specificity toward different TRiC substrates. The isoforms share conserved TRiC binding components while evolving distinct substrate-binding elements that dictate their roles in the TRiC folding environment ( Park et al, 2023 ). PhLPs are exclusive to eukaryotes, and their emergence may correlate with the rise in proteomic complexity.…”

“…The combined use of PhLPs as substrate-specific cochaperones amplifies the capacity of TRiC to fold various substrates. Recent studies have revealed the domain-specific connections between PhLP2A and TRiC, and various functions of the cochaperone in the TRiC network ( Han et al, 2023 , Kelly et al, 2022 , Park et al, 2024 ).

Fig.

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“…During the substrate folding cycle, another cochaperone, such as PhLP2A, orchestrates with TRiC to modulate other cochaperones, or assists substrate folding. Figures are modified and adopted from Park et al (2023) . …”