Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling

Abstract

The TRiC/CCTchaperonin is a 1-MDa hetero-oligomer of 16 subunits that assists the folding of proteins in eukaryotes. Low-resolution structural studies confirmed the TRiC particle to be composed of two stacked octameric rings enclosing a folding cavity. The exact arrangement of the different proteins in the rings underlies the functionality of TRiC and is likely to be conserved across all eukaryotes. Yet despite its importance it has not been determined conclusively, mainly because the different subunits appear nearly identical under low resolution. This work successfully addresses the arrangement problem by the emerging technique of cross-linking, mass spectrometry, and modeling. We cross-linked TRiC under native conditions with a cross-linker that is primarily reactive toward exposed lysine side chains that are spatially close in the context of the particle. Following digestion and mass spectrometry we were able to identify over 60 lysine pairs that underwent crosslinking, thus providing distance restraints between specific residues in the complex. Independently of the cross-link set, we constructed 40,320 (¼8 factorial) computational models of the TRiC particle, which exhaustively enumerate all the possible arrangements of the different subunits. When we assessed the compatibility of each model with the cross-link set, we discovered that one specific model is significantly more compatible than any other model. Furthermore, bootstrapping analysis confirmed that this model is 10 times more likely to result from this cross-link set than the next best-fitting model. Our subunit arrangement is very different than any of the previously reported models and changes the context of existing and future findings on TRiC.group II chaperonins | protein folding | violation distances I n eukaryotes and archaea the folding of nascent and mis-folded polypeptide chains is assisted by a group II chaperonin system known as the thermosome in archea and TRiC (or CCT) in eukaryotes (1). These large protein complexes consist of two stacked octameric rings (2) The open state of the complex binds the polypeptide substrate (4, 5), whereupon closing the substrate is sequestered into a large interior cavity where folding can occur. TRiC has been implicated in the folding pathways of many cytosolic proteins (6), most notably actin and tubulin (7,8).Many archaeal species have just one thermosome gene and a simple homo-oligomeric architecture (9). Other archaeal species have at most three different types of subunits, and there is evidence that the multiple genes are redundant (10). In stark contrast, the eukaryotic TRiC consists of eight different subunit types (CCT1 to CCT8), all of which are essential. The subunit specialization occurred very early in eukaryote evolution (11) and is conserved to such an extent that the sequence identity between mammalian and yeast subunits of the same type is nearly 60%, whereas the sequence identity between the different subunit types in the same organism is only 30%. The diversity of eight distinct subun...

Structures of the Gβ-CCT and PhLP1–Gβ-CCT complexes reveal a mechanism for G-protein β-subunit folding and Gβγ dimer assembly

“…The XL-MS analysis identified a number of intermolecular cross-links between CCT subunits (Dataset S1) that were consistent with the arrangement of the subunits in the CCT ring proposed from previous XL-MS studies (16,17). In addition, (19)] was docked into the mass attributable to Gβ in the cryo-EM reconstruction.…”

Interactions of subunit CCT3 in the yeast chaperonin CCT/TRiC with Q/N-rich proteins revealed by high-throughput microscopy analysis

“…The eight subunits of CCT are arranged in a defined permutation (9), and the orientation of the two rings of CCT with respect to each other is also fixed (10). CCT's hetero-oligomeric structure has enabled a combinatorial mode of protein substrate binding to specific subunits (8,11) to coevolve as found in the case of actin, for example, which binds to particular subunits on both sides of the ring (11).…”

Dynamics, flexibility, and allostery in molecular chaperonins

“…The other Group II chaperonin is found in the cytosol of eukaryotic organisms, and is usually termed CCT (chaperonin containing TCP1) or TRiC (TCP1 ring complex) [4,69]. CCT is composed of eight distinct subunits (CCTa-1, CCTb-2, CCTc-3, CCTd-4, CCTe-5, CCTf-6, CCTg-7 and CCTh-8) organized in a unique intra-and inter-ring arrangement that was recently determined, although the exact hand of the arrangement has not been resolved [70,71].…”

“…Layer 3 (green gradient): the degree of ATP binding and hydrolysis potency in the CCT subunits depicted as ATP++, ATP+ and ATPÀ [99]. Inner layer: cavity electrostatics [70]. and hydrolysis activities are dispensable for the in vivo function of the chaperonin, and generated the most important subunits at the end of the duplication process.…”