4.0-Å resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement

Abstract

The essential double-ring eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) assists the folding of ∼5-10% of the cellular proteome. Many TRiC substrates cannot be folded by other chaperonins from prokaryotes or archaea. These unique folding properties are likely linked to TRiC's unique heterooligomeric subunit organization, whereby each ring consists of eight different paralogous subunits in an arrangement that remains uncertain. Using single particle cryo-EM without imposing symmetry, we determined the mammalian TRiC structure at 4.7-Å resolution. This revealed the existence of a 2-fold axis between its two rings resulting in two homotypic subunit interactions across the rings. A subsequent 2-fold symmetrized map yielded a 4.0-Å resolution structure that evinces the densities of a large fraction of side chains, loops, and insertions. These features permitted unambiguous identification of all eight individual subunits, despite their sequence similarity. Independent biochemical near-neighbor analysis supports our cryo-EM derived TRiC subunit arrangement. We obtained a Cα backbone model for each subunit from an initial homology model refined against the cryo-EM density. A subsequently optimized atomic model for a subunit showed ∼95% of the main chain dihedral angles in the allowable regions of the Ramachandran plot. The determination of the TRiC subunit arrangement opens the way to understand its unique function and mechanism. In particular, an unevenly distributed positively charged wall lining the closed folding chamber of TRiC differs strikingly from that of prokaryotic and archaeal chaperonins. These interior surface chemical properties likely play an important role in TRiC's cellular substrate specificity.asymmetric reconstruction | atomic model | subunit structure

Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro

“…4). The morphology of purified human TRiC was consistent with that of purified TRiC reported in the literature (Cong et al 2010;Dekker et al 2011).…”

“…Chaperonins are divided into two groups: group I (found in prokaryotes and in the chloroplasts and mitochondria of eukaryotes) and group II (found in the Archaeal and eukaryotic cytosol) (Hartl et al 2011). The Archaeal group II chaperonins consist of two seven to nine subunit rings that have one to three different subunits (Bigotti and Clarke 2008), while the eukaryotic group II chaperonin TCP-1 ring complex (TRiC) consists of two identical rings, each with eight different chaperone containing TCP-1 (CCT) subunits (Cong et al 2010).…”

“…Investigations of the arrangement of the CCT subunits in TRiC purified from different species have given conflicting results (Cong et al 2010;Dekker et al 2011). However, a recent novel mass spectrometry method established that bovine TRiC purified from testes tissue and yeast TRiC purified via an interacting protein had the same arrangement with CCT2 and CCT6 forming the homotypic contacts (Leitner et al 2012).…”

Dynamics, flexibility, and allostery in molecular chaperonins

“…Recent years have witnessed a great deal of structural information derived from X-ray and EM data [10,11,[72][73][74][75][76][77][78] (Fig. 1B and C).…”

Homology Modeling: Generating Structural Models to Understand Protein Function and Mechanism

“…Recent advances in CryoEM have led to subnanometer resolutions density maps that can be used to directly refine all-atom structural models [70]. CryoEM densities are usually deposited at the electron microscopy data bank (http://www.emdatabank.…”