Cryo-EM study on the homo-oligomeric ring formation of yeast TRiC/CCT subunits reveals TRiC ring assembly mechanism

Abstract

The complex eukaryotic chaperonin TRiC/CCT helps maintain cellular protein homeostasis, however, its assembly mechanism remains largely unknown. To address the subunit specificity in TRiC assembly, we express each of the individual yeast TRiC subunit in E. coli. Our cryo-EM structural study and biochemical analyses demonstrate that CCT1/2/6 can form TRiC-like homo-oligomeric double ring (HR) complex, however ATP-hydrolysis cannot trigger their ring closure; after deletion of the long N-terminal extension, CCT5 can form the closed double-ring structure; while CCT3/4/7/8 cannot form the HRs. It appears that CCT1 forms a HR in a unique spiral configuration, and ATP-hydrolysis can drive it to re-assemble with an inserted extra subunit-pair. Our data suggest that CCT5 could be the leading subunit in ATP-hydrolysis-driven TRiC ring closure. Moreover, we demonstrate that ADP is sufficient to trigger the assembly of the HRs and TRiC from the assembly intermediate micro-complex form. Our study reveals that through evolution, the more ancestral subunits may have evolved to take more responsibilities in TRiC ring assembly, and we propose a possible assembly mechanism of TRiC involving subunit-pair insertion. Collectively, our study gives hints on the structural basis of subunit specificity in TRiC assembly and cooperativity, beneficial for future TRiC-related therapeutic strategy development.

Pathway and mechanism of tubulin folding mediated by TRiC/CCT conjugated with its ATPase cycle revealed by cryo-EM

“…4a-c). Consistently, previous studies also suggested important roles of CCT3/6/8 in the recognition of other substrates such as mLST8, reovirus σ3 capsid protein, and AML1-175 [26][27][28] . Taken together, the .…”

“…TRiC-mediated substrate folding is closely related to its ATP-driven conformational cycle [22][23][24][25] . TRiC has been proved to display subunit specificity in the complex assembly, ATP consumption and ring closure 16,19,26,27 .…”

Muscle Histopathological Abnormalities in a Patient With a CCT5 Mutation Predicted to Affect the Apical Domain of the Chaperonin Subunit

“…In the last few years, it has been found that, differently from other subunits, CCT5 (and in a less competent manner also the CCT4 subunit) has the ability to form homo-oligomeric complexes (micro-complex and single ring), supporting the hypothesis that single CCT5 homo-oligomeric rings may be the base assembly units for the formation of the functional hetero-oligomeric CCT hexadecamer ( Sergeeva et al, 2019 ; Liu et al, 2021 ). A certain functional ability of the mutant CCT5 in our patient is suggested by its ability to form part of the CCT team, i.e., the CCT hexadecamer.…”

Native mass spectrometry analyses of chaperonin complex TRiC/CCT reveal subunit N-terminal processing and re-association patterns

“…In many paralogs that interface was likely only partly lost as their specific positions in the ring were entrenched, as has been demonstrated for other multimeric complexes with ring topologies 55,56 . Our results could be explained if the residual affinity between paralogs that are not adjacent in TRiC reflects incomplete loss of ancient contacts 7,17,[47][48][49] . Whether they signify moonlighting functions or byproducts of evolution, non-canonical CCT pair contacts must be prevented or displaced during TRiC assembly by kinetic or competitive barriers (Fig.…”

“…Thus, CCT5 can interact with many other CCT subunits even in cells. Recombinant CCT4, CCT1, CCT2, and CCT6 can also homo-oligomerize in the absence of other subunits 48,49 . While we did detect CCT4-CCT4 and CCT2-CCT2 dimers after RP-HPLC, these dimers were not especially abundant in our native MS dissociation analyses.…”