Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling.
The TRiC/CCT chaperonin 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 cross-linking, thus providing distance restraints between specific residues in the complex. Independently of the cross-link set, we constructed 40,320 (= 8 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.
Department of Structural Biology, Stanford University School of Medicine, D100 Fairchild Building, Stanford, CA 94305, USA.
Nir
N
Kalisman
Christopher M
CM
Adams
Michael
M
Levitt
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D056404:Chaperonin Containing TCP-1 / Q000737:chemistry*; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D003432:Cross-Linking Reagents / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism*; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000737:chemistry*; D040681:Structural Homology, Protein
D003432:Cross-Linking Reagents; D021122:Protein Subunits; D056404:Chaperonin Containing TCP-1