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

Nir Kalisman, Christopher M. Adams, Michael Levitt*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

137 Scopus citations

Abstract

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 crosslinking, 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.

Original languageAmerican English
Pages (from-to)2884-2889
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume109
Issue number8
DOIs
StatePublished - 21 Feb 2012
Externally publishedYes

Keywords

  • Group II chaperonins
  • Protein folding
  • Violation distances

Fingerprint

Dive into the research topics of 'Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling'. Together they form a unique fingerprint.

Cite this