Formation of an Intricate Helical Bundle Dictates the Assembly of the 26S Proteasome Lid

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The 26S proteasome is a 2.5 MDa molecular machine that executes the degradation of substrates of the ubiquitin-proteasome pathway. The molecular architecture of the 26S proteasome was recently established by cryo-EM approaches. For a detailed understanding of the sequence of events from the initial binding of polyubiquitylated substrates to the translocation into the proteolytic core complex, it is necessary to move beyond static structures and characterize the conformational landscape of the 26S proteasome. To this end we have subjected a large cryo-EM dataset acquired in the presence of ATP and ATP-γS to a deep classification procedure, which deconvolutes coexisting conformational states. Highly variable regions, such as the density assigned to the largest subunit, Rpn1, are now well resolved and rendered interpretable. Our analysis reveals the existence of three major conformations: in addition to the previously described ATP-hydrolyzing (ATP h ) and ATP-γS conformations, an intermediate state has been found. Its AAA-ATPase module adopts essentially the same topology that is observed in the ATP h conformation, whereas the lid is more similar to the ATP-γS bound state. Based on the conformational ensemble of the 26S proteasome in solution, we propose a mechanistic model for substrate recognition, commitment, deubiquitylation, and translocation into the core particle.conformational switching | proteolysis | proteostasis | quality control I n the ubiquitin-proteasome pathway (UPP) the 26S proteasome performs the degradation of intracellular proteins marked for destruction by the covalent attachment of polyubiquitin chains (1-5). The 2.5 MDa complex consists of the barrel-shaped 20S core particle (CP) as well as one or two copies of the 19S regulatory particle (RP) controlling the entry of substrates into the proteolytic chamber of the CP. The structure of the CP was solved by X-ray crystallography a long time ago (6, 7); whereas, the molecular architecture of the 26S holocomplex was determined only recently using cryo-EM single-particle analysis (SPA) approaches (8-10). The RP comprises a ringshaped AAA-ATPase heterohexamer (Rpt1-6) responsible for substrate unfolding and translocation across the CP gate and 13 RP non-ATPases (Rpn1-3, 5-13, 15) surrounding the AAAATPase module. The role of the Rpns is the acceptance of substrates and their deubiquitylation. For a full mechanistic understanding of the early steps of substrate processing it is essential to reveal its dynamics.The compositional and conformational heterogeneity of 26S proteasome preparations makes the structural characterization of this molecular machine challenging (11). Compositional heterogeneity results from multiple proteins that interact with the 26S proteasome substoichiometrically, such as deubiquitylating enzymes (DUBs) or shuttling ubiquitin (Ub) receptors. Conformational switching of the 26S proteasome is mostly driven by ATP binding and hydrolysis. Each of the six distinct Rpt subunits is able to bind and hydrolyze ATP (12-14), which may...

Section: Distinct 26s Proteasome Conformations At Subnanometer Resolumentioning

confidence: 99%

Deep classification of a large cryo-EM dataset defines the conformational landscape of the 26S proteasome

Unverdorben

Beck

Śledź

et al. 2014

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289

“…Recent cryoelectron microscopy (cryo-EM) analysis of the complete proteasome at moderate resolution (6 to 10 Å) revealed an overall subunit organization of the RP (7)(8)(9)(10)(25)(26)(27)(28). The lid subcomplex, which consists of nine Rpn proteins (Rpn3, Rpn5 to Rpn9, Rpn11, Rpn12, and Dss1/Sem1), exhibits a horseshoe-like architecture and is organized around an elaborate bundle assembled from the C-terminal helices of each subunit but Dss1/Sem1 (20,29). The six Rpt subunits of the base share a general domain organization,…”

mentioning

confidence: 99%

Structural basis for dynamic regulation of the human 26S proteasome

Chen

et al. 2016

159

310

The proteasome is the major engine of protein degradation in all eukaryotic cells. At the heart of this machine is a heterohexameric ring of AAA (ATPases associated with diverse cellular activities) proteins that unfolds ubiquitylated target proteins that are concurrently translocated into a proteolytic chamber and degraded into peptides. Using cryoelectron microscopy, we determined a near–atomic-resolution structure of the 2.5-MDa human proteasome in its ground state, as well as subnanometer-resolution structures of the holoenzyme in three alternative conformational states. The substrate-unfolding AAA-ATPase channel is narrowed by 10 inward-facing pore loops arranged into two helices that run in parallel with each other, one hydrophobic in character and the other highly charged. The gate of the core particle was unexpectedly found closed in the ground state and open in only one of the alternative states. Coordinated, stepwise conformational changes of the regulatory particle couple ATP hydrolysis to substrate translocation and regulate gating of the core particle, leading to processive degradation.

“…The nine-subunit lid is thought to self-assemble without dedicated chaperone proteins (28)(29)(30)(31), and then joins with the base to form the RP. Based on biochemical and proteomic identification of assembly intermediates, the base and lid appear to be assembled via distinct precursor complexes, which do not overlap in their subunit composition (9-14, 16, 28, 29).…”

mentioning

confidence: 99%

Nucleotide-dependent switch in proteasome assembly mediated by the Nas6 chaperone

Tian

Langager

et al. 2017

The proteasome is assembled via the nine-subunit lid, nine-subunit base, and 28-subunit core particle (CP). Previous work has shown that the chaperones Rpn14, Nas6, Hsm3, and Nas2 each bind a specific ATPase subunit of the base and antagonize base-CP interaction. Here, we show that the Nas6 chaperone also obstructs base-lid association. Nas6 alternates between these two inhibitory modes according to the nucleotide state of the base. When ATP cannot be hydrolyzed, Nas6 interferes with base-lid, but not base-CP, association. In contrast, under conditions of ATP hydrolysis, Nas6 obstructs base-CP, but not base-lid, association. Modeling of Nas6 into cryoelectron microscopy structures of the proteasome suggests that Nas6 controls both base-lid affinity and base-CP affinity through steric hindrance; Nas6 clashes with the lid in the ATP-hydrolysis-blocked proteasome, but clashes instead with the CP in the ATP-hydrolysis-competent proteasome. Thus, Nas6 provides a dual mechanism to control assembly at both major interfaces of the proteasome.proteasome | chaperone | AAA + ATPase | assembly | Nas6