The 26S proteasome is at the executive end of the ubiquitinproteasome pathway for the controlled degradation of intracellular proteins. While the structure of its 20S core particle (CP) has been determined by X-ray crystallography, the structure of the 19S regulatory particle (RP), which recruits substrates, unfolds them, and translocates them to the CP for degradation, has remained elusive. Here, we describe the molecular architecture of the 26S holocomplex determined by an integrative approach based on data from cryoelectron microscopy, X-ray crystallography, residue-specific chemical cross-linking, and several proteomics techniques. The "lid" of the RP (consisting of Rpn3/5/6/7/8/9/11/12) is organized in a modular fashion. Rpn3/5/6/7/9/12 form a horseshoe-shaped heterohexamer, which connects to the CP and roofs the AAAATPase module, positioning the Rpn8/Rpn11 heterodimer close to its mouth. Rpn2 is rigid, supporting the lid, while Rpn1 is conformationally variable, positioned at the periphery of the ATPase ring. The ubiquitin receptors Rpn10 and Rpn13 are located in the distal part of the RP, indicating that they were recruited to the complex late in its evolution. The modular structure of the 26S proteasome provides insights into the sequence of events prior to the degradation of ubiquitylated substrates.coiled coils | mass spectrometry | proteasome-COP9-eIF3 domain | proteasome-cyclosome repeats I n eukaryotes, the ubiquitin-proteasome pathway (UPP) is essential for proteostasis: Misfolded proteins or otherwise defective proteins as well as short-lived regulatory proteins are eliminated by degradation (1). The UPP regulates many fundamental cellular processes, such as protein quality control, DNA repair, and signal transduction (for review see ref.2). The 26S proteasome is the most downstream element of the UPP, executing the degradation of polyubiquitylated substrates (3-5). It consists of the barrelshaped core particle (CP; approximately 700 kDa), which sequesters the proteolytically active site in its central cavity, and the regulatory particle (RP; approximately 900 kDa), which is attached at either one or both ends of the CP and prepares substrates for degradation (6).The RP consists of 19 different canonical subunits, including six regulatory particle AAA-ATPase subunits (Rpt1-6) and 13 regulatory particle non-ATPase subunits (Rpn1-3, Rpn5-13, and Rpn15). The integral ubiquitin (Ub) receptors Rpn10 and Rpn13 recognize polyubiquitylated substrates (7-9). Alternatively, polyubiquitylated substrates can be recruited by shuttling Ub-receptors (Dsk2, Rad23, Ddi2), which bind to substrates with their Ub-associated domain, and to Rpn1, Rpn10, or Rpn13 at their Ub-like domain (5). The metalloprotease Rpn11 deubiquitylates substrates prior to their degradation (10, 11). The functions of the other Rpn subunits remain to be established. The AAA-ATPases form a hexameric ring that unfolds substrates, opens the gate to the CP, and eventually translocates the substrates to the CP.Electron microscopy (EM) (12) and X-...
The 26S proteasome operates at the executive end of the ubiquitinproteasome pathway. Here, we present a cryo-EM structure of the Saccharomyces cerevisiae 26S proteasome at a resolution of 7.4 Å or 6.7 Å (Fourier-Shell Correlation of 0.5 or 0.3, respectively). We used this map in conjunction with molecular dynamics-based flexible fitting to build a near-atomic resolution model of the holocomplex. The quality of the map allowed us to assign α-helices, the predominant secondary structure element of the regulatory particle subunits, throughout the entire map. We were able to determine the architecture of the Rpn8/Rpn11 heterodimer, which had hitherto remained elusive. The MPN domain of Rpn11 is positioned directly above the AAA-ATPase N-ring suggesting that Rpn11 deubiquitylates substrates immediately following commitment and prior to their unfolding by the AAA-ATPase module. The MPN domain of Rpn11 dimerizes with that of Rpn8 and the C-termini of both subunits form long helices, which are integral parts of a coiled-coil module. Together with the C-terminal helices of the six PCI-domain subunits they form a very large coiled-coil bundle, which appears to serve as a flexible anchoring device for all the lid subunits.protein degradation | electron microscopy | deubiquitylating enzyme T he 26S proteasome is a 2.5 MDa molecular machine designed for the controlled degradation of proteins marked for destruction by the covalent attachment of polyubiquitin chains [for reviews see (1-3)]. It is composed of two copies, each of 33 canonical subunits, as well as some proteasome interacting proteins (PIPs). The 26S holocomplex comprises two types of subcomplexes: the cylindrical 20S core particle (CP) harbouring the proteolytic chamber and the two 19S regulatory particles (RPs), which attach to opposite ends of CP cylinder. The RPs have multiple roles in preparing substrates for degradation: They recognize and bind ubiquitylated proteins, they deubiquitylate them followed by their unfolding, and they control the opening of the gate which gives access to the interior of the CP.While the structure of the 20S core complex was determined by X-ray crystallography almost two decades ago (4, 5), the structure of the 26S complex remained recalcitrant to crystallization attempts, presumably due to its conformational and compositional heterogeneity. Recently, the subunit architecture of the holocomplex has been determined by cryo-electron microscopy (EM) single particle analysis (SPA and ref. 6, 7) independently by two groups using different approaches for the assignment of RP subunits. Lander, et al. (6) obtained a 9 Å resolution map (Fouriershell correlation, FSC ¼ 0.5) of the 26S Saccharomyces cerevisiae proteasome and they determined the subunit positions by means of fusion constructs and automated segmentation methods. Lasker, et al. (7) performed an exhaustive computational search of possible subunit configurations within the boundaries of an 8.5 Å map of the 26S proteasome from Schizosaccharomyces pombe scoring possible configurati...
The study of proteins and protein complexes using chemical crosslinking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches. P roteins exert the majority of their functions in the form of protein complexes to control cellular signaling, protein synthesis, folding and degradation, and many more essential processes. Therefore, elucidating the composition and structure of such complexes has been a longstanding goal of biological research.MS-based proteomics has emerged as one of the main techniques to identify and quantify proteins and their modifications in biological samples such as isolated complexes, proteome fractions, or whole proteomes. Various MS methods now provide structural information on protein assemblies (1-3). Among them, chemical cross-linking and identification of cross-linked peptides by MS (XL-MS) has been increasingly applied to determine the subunit arrangements of biologically relevant complexes (4-6). Such XL-MS experiments indicate the locations of cross-linking sites and thus the spatial proximity of reactive groups that are connected by a covalent bond. This information is then used to determine the positioning of subunits or locate interacting regions, alone or in combination with other techniques such as NMR spectroscopy, electron microscopy, and X-ray crystallography.In the last few years, optimized protocols and new computational tools for the reliable analysis of XL-MS datasets resulted in significant advances of the XL-MS technology (4-6). These advances have contributed to the emergence of a robust, integrated XL-MS method that has been successfully applied for structure determination of a number of large protein complexes (7-11) and the detection of direct, physical interactions in whole cells (12)(13)(14). To date, the cross-linking chemistries applied in these studies have targ...
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...
The 26S proteasome is a 2.5-MDa, ATP-dependent multisubunit proteolytic complex that processively destroys proteins carrying a degradation signal. The proteasomal ATPase heterohexamer is a key module of the 19S regulatory particle; it unfolds substrates and translocates them into the 20S core particle where degradation takes place. We used cryoelectron microscopy single-particle analysis to obtain insights into the structural changes of 26S proteasome upon the binding and hydrolysis of ATP. The ATPase ring adopts at least two distinct helical staircase conformations dependent on the nucleotide state. The transition from the conformation observed in the presence of ATP to the predominant conformation in the presence of ATP-γS induces a sliding motion of the ATPase ring over the 20S core particle ring leading to an alignment of the translocation channels of the ATPase and the core particle gate, a conformational state likely to facilitate substrate translocation. Two types of intersubunit modules formed by the large ATPase domain of one ATPase subunit and the small ATPase domain of its neighbor exist. They resemble the contacts observed in the crystal structures of ClpX and proteasome-activating nucleotidase, respectively. The ClpX-like contacts are positioned consecutively and give rise to helical shape in the hexamer, whereas the proteasome-activating nucleotidase-like contact is required to close the ring. Conformational switching between these forms allows adopting different helical conformations in different nucleotide states. We postulate that ATP hydrolysis by the regulatory particle ATPase (Rpt) 5 subunit initiates a cascade of conformational changes, leading to pulling of the substrate, which is primarily executed by Rpt1, Rpt2, and Rpt6.AAA ATPase | ubiquitin-proteasome pathway | hybrid methods in structural biology T he 26S proteasome is the executive arm of the ubiquitin-proteasome system (UPS), the major pathway for intracellular protein degradation in eukaryotic cells (1, 2). It degrades proteins that are marked for destruction by the covalent attachment of a polyubiquitin chain. The 2.5-MDa complex comprises a core particle (CP), the 20S proteasome, harboring the proteolytically active sites and one or two regulatory particles (RPs), which associate with the barrel-shaped CP. A key component of the RP is the AAA-ATPase module (ATPase associated with various cellular activities), which associates with the α-rings of the CP and prepares substrates for degradation. Whereas the 26S proteasome is the only soluble ATP-dependent protease in the eukaryotic cytosol, bacteria possess several different ATPdependent proteases like the ClpXP, HslUV, and the eubacterial proteasome-ARC/ Mpa (AAA ATPase forming ring-shaped complexes/mycobacterial proteasomal ATPase) system (3).The CP consists of four seven-membered rings (4). The two adjacent heteroheptameric β-rings form a cavity, which harbors the active sites (5, 6). The β-rings are sandwiched between two heteroheptameric α-rings, whose C-termini form a gate con...
In mammalian cells, secretory and membrane proteins are translocated across or inserted into the endoplasmic reticulum (ER) membrane by the universally conserved protein-conducting channel Sec61, which has been structurally studied in isolated, detergent-solubilized states. Here we structurally and functionally characterize native, non-solubilized ribosome-Sec61 complexes on rough ER vesicles using cryo-electron tomography and ribosome profiling. Surprisingly, the 9-Å resolution subtomogram average reveals Sec61 in a laterally open conformation, even though the channel is not in the process of inserting membrane proteins into the lipid bilayer. In contrast to recent mechanistic models for polypeptide translocation and insertion, our results indicate that the laterally open conformation of Sec61 is the only conformation present in the ribosome-bound translocon complex, independent of its functional state. Consistent with earlier functional studies, our structure suggests that the ribosome alone, even without a nascent chain, is sufficient for lateral opening of Sec61 in a lipid environment.
Proteasomes execute the degradation of most cellular proteins. Although the 20S core particle (CP) has been studied in great detail, the structure of the 19S regulatory particle (RP), which prepares ubiquitylated substrates for degradation, has remained elusive. Here, we report the crystal structure of one of the RP subunits, Rpn6, and we describe its integration into the cryo-EM density map of the 26S holocomplex at 9.1 Å resolution. Rpn6 consists of an α-solenoid-like fold and a proteasome COP9/signalosome eIF3 (PCI) module in a right-handed suprahelical configuration. Highly conserved surface areas of Rpn6 interact with the conserved surfaces of the Pre8 (alpha2) and Rpt6 subunits from the alpha and ATPase rings, respectively. The structure suggests that Rpn6 has a pivotal role in stabilizing the otherwise weak interaction between the CP and the RP.
The 26S proteasome is a 2.5 MDa molecular machine for the degradation of substrates of the ubiquitin-proteasome pathway with a key role in cellular proteostasis. Until recently, only the structure of its core particle, the 20S proteasome, could be studied in detail, whereas the 19S regulatory particle or the holocomplex remained elusive. Novel integrative approaches have now revealed the molecular architecture of the entire complex and provided the first insights into the conformational changes during its functional cycle. Here we review the problems in structural studies of the 26S proteasome, the methods that made possible its structure determination, the architectural principles of the holocomplex, and its conformational space. These advances provide valuable insights into the mechanism of substrate recruitment and processing preceding their destruction in the 20S core particle.
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