TRiC folds the giant ciliary protein IFT172 via a non-canonical open-state mechanism

This study reveals that the eukaryotic chaperonin TRiC folds the oversized ciliary protein IFT172 through a non-canonical "fold-and-eject" mechanism involving chamber expansion and concurrent HSP70 assistance, challenging the classical view of the closed chamber as an obligate folding cage and establishing a new paradigm for ciliary biogenesis.

The Gist

Imagine your cell is a bustling city, and inside this city, there are massive construction projects happening. One of the most important projects is building cilia—tiny, hair-like antennas that stick out of cells to help them sense the world and move fluids. To build these antennas, the cell needs a giant piece of machinery called IFT172.

But here's the problem: IFT172 is a giant. It's about 200,000 units of weight (kDa), which is way too big to fit inside the cell's standard "folding factory."

The Problem: A Sofa That Won't Fit in the Elevator

For decades, scientists believed the cell's main folding machine, called TRiC, worked like a folding elevator.

1. You drop a protein (the "sofa") into the elevator.
2. The doors close.
3. Inside the dark, enclosed box, the sofa gets folded into its perfect shape.
4. The doors open, and the folded sofa comes out.

This works great for small sofas (small proteins). But IFT172 is like a king-size sectional sofa. If you try to put it in the elevator, it won't fit. The doors can't close. For years, scientists were stuck: How does the cell fold this giant thing if the elevator is too small?

The Discovery: A "Divide and Conquer" Strategy

This paper reveals that the cell has a brilliant, new way of handling these giants. Instead of trying to force the whole sofa into the elevator, the cell uses a two-team strategy:

1. The Elevator Team (TRiC):
The TRiC machine grabs the front part of the giant sofa (the N-terminal WD40 domains) and pulls just that part inside its chamber.

• The Twist: To make room, the elevator doesn't just close; it stretches. Specific parts of the elevator doors bend outward in a "Z-shape," widening the opening just enough to let the bulky front in, while the rest of the sofa sticks out the back.

2. The Ground Crew (HSP70):
While the front is inside the elevator, the back part of the sofa (the C-terminal TPR domain) is still sticking out into the room. A different helper, called HSP70, grabs this sticking-out part and holds it steady so it doesn't get tangled or knotted.

The Analogy: Imagine two people trying to fold a massive, tangled sleeping bag. One person holds the top half inside a small tent (TRiC), while the other person stands outside holding the bottom half straight (HSP70). They work together, but on different parts of the same object.

The Surprise: Folding Before the Doors Close

The most surprising discovery in this paper is about when the folding happens.

• Old Belief: The protein must be fully enclosed in the closed elevator to fold.
• New Reality: The front part of IFT172 actually gets folded while the elevator doors are still open and the machine is powered up (ATP-bound).

Think of it like a magic trick. The sofa gets shaped perfectly while it's still half-in, half-out of the box. Once that front part is folded, the elevator doors do close, but they don't trap the sofa. Instead, the closing doors act like a spring-loaded launcher, shooting the now-folded sofa out the other side.

The authors call this a "Fold-and-Eject" mechanism.

• Open State: The folding happens here.
• Closing State: The doors slam shut to push the finished product out, not to keep it in.

Why This Matters

If this system breaks, the giant sofa (IFT172) never gets built correctly. The cell's antennas (cilia) fail to form or function. This leads to serious human diseases called ciliopathies, which can affect vision, kidney function, and brain development.

The Big Picture

This paper changes how we understand the cell's machinery. It shows that:

1. Flexibility is key: The folding machine isn't a rigid box; it's a stretchy, adaptable tool that can reshape itself to fit huge objects.
2. Teamwork: Different helpers (TRiC and HSP70) can work on the same giant protein at the same time, but on different sections.
3. New Rules: Sometimes, you don't need to lock the doors to get the job done; in fact, locking the doors might be the signal to finish and release the work.

In short, the cell has evolved a clever, "divide-and-conquer" way to fold its biggest, most complex proteins, ensuring that the tiny antennas on our cells can keep working so we can see, smell, and move properly.

Technical Summary

1. Problem Statement

The eukaryotic chaperonin TRiC/CCT is essential for folding approximately 10% of the cytosolic proteome, including cytoskeletal proteins and WD40 $\beta$-propeller proteins. The canonical model of TRiC function posits an ATP-driven cycle where the substrate is encapsulated within a closed chamber (~70 kDa capacity) to facilitate folding via steric confinement.

However, a longstanding paradox exists: TRiC is required for the biogenesis of massive, multi-domain proteins that far exceed the volumetric capacity of its closed cavity. A prime example is IFT172, the largest subunit (~200 kDa) of the Intraflagellar Transport (IFT) machinery, which contains tandem N-terminal WD40 $\beta$-propellers and a C-terminal TPR solenoid. It was unclear how TRiC accommodates such oversized clients, whether it coordinates with other chaperones, and at which stage of the ATPase cycle productive folding occurs.

2. Methodology

The authors employed an integrative structural and functional approach:

• Biochemical Characterization: Affinity purification of FLAG-tagged IFT172 from HEK293F cells followed by mass spectrometry (MS) to identify interactors. Co-immunoprecipitation (Co-IP) and glycerol gradient centrifugation validated complex formation.
• Cryo-Electron Microscopy (Cryo-EM): High-resolution structures were determined for:
  • The apo-state TRiC-IFT172 complex (3.55 Å).
  • The ATP-AlFx-bound (transition state) complex, capturing both open and closed ring conformations.
  • 3D Variability Analysis (3DVA) was used to map conformational dynamics.
• Cross-linking Mass Spectrometry (XL-MS): Used to map interaction interfaces between IFT172, TRiC subunits, and the co-chaperone HSP70. Distance restraints were integrated with AlphaFold3 predictions to model binding modes.
• In Vivo Functional Assays: Caenorhabditis elegans models were used. CRISPR/Cas9 generated endogenous IFT172::GFP strains. Neuron-specific RNAi knockdown of TRiC subunits (cct-5 and cct-8) was performed to assess impacts on ciliary morphology and IFT dynamics via live-cell imaging and kymograph analysis.

3. Key Contributions & Results

A. A "Divide-and-Conquer" Chaperone Strategy

The study reveals that TRiC does not act alone. Instead, it employs a spatially segregated "divide-and-conquer" mechanism:

• TRiC Engagement: TRiC specifically captures the N-terminal WD40 domains (WD40-1 and WD40-2) of IFT172 within its central chamber.
• HSP70 Engagement: The chaperone HSP70 binds independently to the C-terminal TPR solenoid, which remains exposed in the cytosol.
• Coordination: There is no direct stable complex between TRiC and HSP70; rather, they simultaneously stabilize distinct domains of the same polypeptide, preventing aggregation of the massive substrate.

B. Allosteric Remodeling of the TRiC Chamber

To accommodate the ~200 kDa substrate, TRiC undergoes dramatic structural plasticity:

• Z-Shaped Bending: Specific subunits (CCT2, CCT4, and CCT7) undergo a pronounced Z-shaped outward bending of their apical (A) and intermediate (I) domains.
• Chamber Expansion: This remodeling expands the chamber aperture by up to 38.8 Å, creating the steric clearance necessary for the bulky substrate to enter while allowing the TPR domain to protrude.
• Stabilization: These bent conformations are stabilized by electrostatic networks between the bent subunits and their neighbors.

C. The "Fold-and-Eject" Mechanism (Non-Canonical Pathway)

The most significant finding challenges the "encapsulation dogma":

• Folding in the Open State: In the ATP-bound open state, the WD40-1 domain of IFT172 reaches a near-native, folded conformation before ring closure. This folding is mediated by electrostatic interactions with the flexible, disordered N- and C-terminal tails of TRiC subunits, rather than rigid cavity walls.
• Ejection upon Closure: Upon ATP hydrolysis and ring closure, the chamber does not encapsulate the substrate. Instead, the closed state is empty of IFT172 (often occupied by endogenous tubulin). The transition to the closed state acts as a mechanical trigger for substrate ejection.
• Conclusion: Productive folding occurs in the open, ATP-bound chamber, and closure serves to release the folded product, not to contain it.

D. In Vivo Relevance and Disease Link

• Ciliopathy Connection: Mutations in IFT172 associated with ciliopathies (e.g., Bardet-Biedl syndrome, Jeune syndrome) were tested. The severe I411N mutation, located in the WD40-2 domain, significantly impaired TRiC binding, linking defective chaperonin recognition directly to disease pathogenesis.
• Physiological Necessity: In C. elegans, partial depletion of TRiC subunits did not abolish ciliogenesis (cilia length remained normal) but caused a severe defect in IFT transport dynamics. This confirms that TRiC is essential for the functional assembly of the IFT machinery, not just its structural formation.
• Conservation: The study identifies a conserved WD40-TPR architecture in five other IFT subunits (IFT121, IFT122, IFT140, IFT144, IFT80), all of which interact with TRiC, suggesting this mechanism is a general paradigm for ciliary protein biogenesis.

4. Significance

• Redefining Chaperonin Mechanisms: The paper overturns the classical view that the closed chamber is an obligate folding cage for all TRiC substrates. It establishes a new "fold-and-eject" paradigm for oversized proteins.
• Structural Plasticity: It demonstrates that TRiC possesses intrinsic, large-scale conformational flexibility (symmetry breaking) to adapt to diverse client sizes, a feature distinct from the rigid GroEL-GroES system.
• Proteostasis Hub: TRiC is identified as a central hub for ciliary biogenesis, coordinating with HSP70 to fold giant multi-domain proteins.
• Clinical Implications: The findings provide a mechanistic basis for ciliopathies caused by mutations that disrupt chaperone recognition rather than the protein's native function, opening new avenues for therapeutic intervention in protein folding diseases.

In summary, this work resolves the "size paradox" of TRiC folding by revealing a dynamic, cooperative, and non-canonical pathway where structural remodeling and open-state folding allow the chaperonin to service the most complex proteins in the eukaryotic proteome.