Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a Reprint requests to: Kevin W. Plaxco, Department of Chemistry and Biochemistry, University of California, Santa Barbara, Santa Barbara, CA 93106, USA; e-mail: kwp@chem.ucsb.edu; fax: (805) 893-4120.Abbreviations: GuHCl, guanidine hydrochloride; tris, tris hydroxymethylaminoethane; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; TCEP, tris(2-carboxyethyl)phosphine; CD, circular dichroism. Article published online ahead of print. Article and publication date are at
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
The ring-shaped hetero-oligomeric chaperonin TRiC/CCT uses ATP to fold a diverse subset of eukaryotic proteins. To define the basis of TRiC/CCT substrate recognition, we mapped the chaperonin interactions with the VHL tumor suppressor. VHL has two well-defined TRiC binding determinants. Each determinant contacts a specific subset of chaperonin subunits, indicating that TRiC paralogs exhibit distinct but overlapping specificities. The substrate binding site in these subunits localizes to a helical region in the apical domains that is structurally equivalent to that of bacterial chaperonins. Transferring the distal portion of helix 11 between TRiC subunits suffices to transfer specificity for a given substrate motif. We conclude that the architecture of the substrate binding domain is evolutionarily conserved among eukaryotic and bacterial chaperonins. The unique combination of specificity and plasticity in TRiC substrate binding may diversify the range of motifs recognized by this chaperonin and contribute to its unique ability to fold eukaryotic proteins.
In order to operate in a coordinated fashion, multisubunit enzymes use cooperative interactions intrinsic to their enzymatic cycle, but this process remains poorly understood. Accordingly, ATP number distributions in various hydrolyzed states have been obtained for single copies of the mammalian double-ring multisubunit chaperonin TRiC/CCT in free solution using the emission from chaperoninbound fluorescent nucleotides and closed-loop feedback trapping provided by an Anti-Brownian ELectrokinetic trap. Observations of the 16-subunit complexes as ADP molecules are dissociating shows a peak in the bound ADP number distribution at 8 ADP, whose height falls over time with little shift in the position of the peak, indicating a highly cooperative ADP release process which would be difficult to observe by ensemble-averaged methods. When AlFx is added to produce ATP hydrolysis transition state mimics (ADP·AlFx) locked to the complex, the peak at 8 nucleotides dominates for all but the lowest incubation concentrations. Although ensemble averages of the single-molecule data show agreement with standard cooperativity models, surprisingly, the observed number distributions depart from standard models, illustrating the value of these single-molecule observations in constraining the mechanism of cooperativity. While a complete alternative microscopic model cannot be defined at present, the addition of subunit-occupancy-dependent cooperativity in hydrolysis yields distributions consistent with the data.single molecule | allostery | fluorescence | enzymology | nucleotide counting Y ears of study of cooperativity for proteins with multiple ligand binding sites have yielded important insights, ever since the role of cooperative oxygen binding to hemoglobin in high oxygendelivery throughput was recognized (1-3). Multisubunit enzymes usually hydrolyze ATP in a concerted fashion, but actually observing this process, enzyme by enzyme, can provide a deeper picture of the underlying cooperativity resulting from communication among the various subunits. A well known example has been the study of the rotary motor F1-ATPase by single-molecule techniques (4). Another class of multisubunit cellular machines is the double-ring type I and type II chaperonins which can have from 14 to 18 subunits each of which can hydrolyze ATP (5, 6). Ensemble studies of the bacterial type I GroEL/GroES system, for example, have indicated that ATP binds subunits in one 7-membered ring with positive cooperativity, while negative cooperativity operates between rings (7). We focus on the cooperative process operative in the mammalian type II chaperonin TRiC/CCT, which has two ring-shaped cavities with built-in lids composed of eight different subunits each. TRiC is essential for the folding of a number of key proteins in mammalian cells, including actin, tubulin, and many cell cycle regulators (8). Previous ensemble measurements of ATP-induced allosteric transition rates and steady-state ATPase rate in TRiC show evidence for positive and negative cooperativity du...
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