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...
In eukaryotic cells, the ubiquitin-proteasome system (UPS) is responsible for the regulated degradation of intracellular proteins. The 26S holocomplex comprises the core particle (CP), where proteolysis takes place, and one or two regulatory particles (RPs). The base of the RP is formed by a heterohexameric AAA + ATPase module, which unfolds and translocates substrates into the CP. Applying single-particle cryo-electron microscopy (cryo-EM) and image classification to samples in the presence of different nucleotides and nucleotide analogs, we were able to observe four distinct conformational states (s1 to s4). The resolution of the four conformers allowed for the construction of atomic models of the AAA + ATPase module as it progresses through the functional cycle. In a hitherto unobserved state (s4), the gate controlling access to the CP is open. The structures described in this study allow us to put forward a model for the 26S functional cycle driven by ATP hydrolysis.26S proteasome | cryo-electron microscopy | AAA + ATPase | integrative modeling | single-particle analysis I n eukaryotic cells, the ubiquitin-proteasome system (UPS) degrades proteins that are misfolded, damaged, or no longer needed (1). The 26S proteasome is a 2.5-MDa multisubunit complex comprising the barrel-shaped 20S core particle (CP), where degradation takes place, and one or two 19S regulatory particles (RPs), which bind to the ends of the CP (2-4). The CP is built of four coaxially stacked heteroheptameric rings of α-and β-subunits in the order of αββα (5). Three of the seven β-subunits are catalytically active; substrates are sequestered from the cellular environment in a chamber formed by the two β-rings (6, 7). This self-compartmentalization is a hallmark of many intracellular proteases (8). Substrate access to the proteolytic chamber is controlled by the α-subunit N-terminal extensions, forming a gate (3). Most of proteasome activators, including the RP, contain C-terminal hydrophobic-tyrosine-X (HbYX) motifs, which have been reported to insert into α-ring pockets, triggering gate opening (9-11).The RP is composed of at least 19 canonical subunits and interacts substoichiometrically with an array of proteasomeinteracting proteins that modulate RP function (3). The RP is divided into the "base" and the "lid" subcomplexes. The core of the base is formed by a heterohexameric ATPase associated with various cellular activities (AAA + ATPase), which is the driver of large-scale conformational dynamics of the RP. The AAA + ATPase prepares substrates for degradation in coordination with at least three ubiquitin receptors [26S proteasome non-ATPase regulatory subunit 1 (Rpn1), Rpn10, and Rpn13] (12-14) and a deubiquitylating subunit (Rpn11) (15, 16). Other subunits have structural roles, such as holding the CP and RP together, or in coordinating the movements needed to position the substrates above the pore of the AAA + ATPase for unfolding and translocation (17, 18). The AAA + ATPase is lined by aromatic-hydrophobic loops (pore-1 loops), wh...
SUMMARY The proteasome is the central protease for intracellular protein breakdown. Coordinated binding and hydrolysis of ATP by the six proteasomal ATPase subunits induces conformational changes that drive the unfolding and translocation of substrates into the proteolytic 20S core particle for degradation. Here, we combine genetic and biochemical approaches with cryo-electron microscopy and integrative modeling to dissect the relationship between individual nucleotide binding events and proteasome conformational dynamics. We demonstrate unique impacts of ATP binding by individual ATPases on the proteasome conformational distribution and report two conformational states of the proteasome suggestive of a rotary ATP hydrolysis mechanism. These structures, coupled with functional analyses, reveal key roles for the ATPases Rpt1 and Rpt6 in gating substrate entry into the core particle. This deepened knowledge of proteasome conformational dynamics reveals key elements of intersubunit communication within the proteasome and clarifies the regulation of substrate entry into the proteolytic chamber.
Protein degradation in eukaryotic cells is performed by the UbiquitinProteasome System (UPS). The 26S proteasome holocomplex consists of a core particle (CP) that proteolytically degrades polyubiquitylated proteins, and a regulatory particle (RP) containing the AAA-ATPase module. This module controls access to the proteolytic chamber inside the CP and is surrounded by non-ATPase subunits (Rpns) that recognize substrates and deubiquitylate them before unfolding and degradation. The architecture of the 26S holocomplex is highly conserved between yeast and humans. The structure of the human 26S holocomplex described here reveals previously unidentified features of the AAA-ATPase heterohexamer. One subunit, Rpt6, has ADP bound, whereas the other five have ATP in their binding pockets. Rpt6 is structurally distinct from the other five Rpt subunits, most notably in its pore loop region. For Rpns, the map reveals two main, previously undetected, features: the C terminus of Rpn3 protrudes into the mouth of the ATPase ring; and Rpn1 and Rpn2, the largest proteasome subunits, are linked by an extended connection. The structural features of the 26S proteasome observed in this study are likely to be important for coordinating the proteasomal subunits during substrate processing.proteostasis | cryo-electron microscopy | AAA-ATPase | integrative modeling T he 26S proteasome is an ATP-dependent multisubunit protease degrading polyubiquitylated proteins (1, 2). It operates at the executive end of the Ubiquitin-Proteasome System (UPS) and has a key role in cellular proteostasis. The 26S proteasome selectively removes misfolded proteins or proteins no longer needed and it is critically involved in numerous cellular processes such as protein quality control, regulation of metabolism, cell cycle control, or antigen presentation. Malfunctions of the UPS are associated with various pathologies, including neurodegenerative diseases and cancer. Therefore, the proteasome is an important pharmaceutical target, and a high-resolution structure is a prerequisite for structure-based drug design (3).The 26S proteasome comprises the 20S cylindrical core particle (CP), where proteolysis takes place, and 19S regulatory particles (RPs). In cellular environments, 26S holocomplexes with either one or two RPs bound to the ends of the cylindershaped CP coexist (4). The role of the RPs is to recruit ubiquitylated substrates, to cleave off their polyubiquitin tags, and to unfold and translocate them into the CP for degradation into short peptides. Whereas X-ray crystallography has revealed the atomic structures first of archaeal 20S proteasome (5) and subsequently of the yeast (6) and mammalian proteasome (7), only lower-resolution structures were available for the 26S holocomplex. Given the compositional and conformational heterogeneity of the RP, single-particle cryo-electron microscopy (cryo-EM) has been the most successful approach to determining the structure of the 26S holocomplex (8). At this point, the most detailed insights have been obtained ...
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