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...

The 26S proteasome is a key player in eukaryotic protein quality control and in the regulation of numerous cellular processes. Here, we describe quantitative in situ structural studies of this highly dynamic molecular machine in intact hippocampal neurons. We used electron cryotomography with the Volta phase plate, which allowed high fidelity and nanometer precision localization of 26S proteasomes. We undertook a molecular census of single- and double-capped proteasomes and assessed the conformational states of individual complexes. Under the conditions of the experiment—that is, in the absence of proteotoxic stress—only 20% of the 26S proteasomes were engaged in substrate processing. The remainder was in the substrate-accepting ground state. These findings suggest that in the absence of stress, the capacity of the proteasome system is not fully used.

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 ...

In eukaryotic cells, the 26S proteasome is responsible for the regulated degradation of intracellular proteins. Several cofactors interact transiently with this large macromolecular machine and modulate its function. The deubiquitylating enzyme ubiquitin C-terminal hydrolase 6 [Ubp6; ubiquitin-specific protease (USP) 14 in mammals] is the most abundant proteasome-interacting protein and has multiple roles in regulating proteasome function. Here, we investigate the structural basis of the interaction between Ubp6 and the 26S proteasome in the presence and absence of the inhibitor ubiquitin aldehyde. To this end we have used single-particle electron cryomicroscopy in combination with cross-linking and mass spectrometry. Ubp6 binds to the regulatory particle non-ATPase (Rpn) 1 via its N-terminal ubiquitin-like domain, whereas its catalytic USP domain is positioned variably. Addition of ubiquitin aldehyde stabilizes the binding of the USP domain in a position where it bridges the proteasome subunits Rpn1 and the regulatory particle triple-A ATPase (Rpt) 1. The USP domain binds to Rpt1 in the immediate vicinity of the Ubp6 active site, which may effect its activation. The catalytic triad is positioned in proximity to the mouth of the ATPase module and to the deubiquitylating enzyme Rpn11, strongly implying their functional linkage. On the proteasome side, binding of Ubp6 favors conformational switching of the 26S proteasome into an intermediateenergy conformational state, in particular upon the addition of ubiquitin aldehyde. This modulation of the conformational space of the 26S proteasome by Ubp6 explains the effects of Ubp6 on the kinetics of proteasomal degradation.conformational switching | proteolysis | proteostasis | quality control D egradation of proteins that are misfolded, damaged, or no longer needed is an essential element of cellular homeostasis. In eukaryotic cells, the ubiquitin-proteasome system (UPS) is the major pathway for regulated protein degradation (1). Proteins that are processed by the UPS are marked for destruction by polyubiquitin chains, which are recognized as a degradation signal by the 26S proteasome.The 26S proteasome consists of the core particle (CP), which degrades substrates into short peptides, and one or two 19S regulatory particles (RP), which associate with the ends of the cylinder-shaped CP to recruit substrates and prepare them for degradation (2, 3). Although the structure of the CP has been known for more than two decades (4, 5), the molecular architecture of the RP was unraveled by cryo-electron microscope (EM)-based approaches only recently (6-9). It comprises six RP triple A (AAA) ATPases (Rpt), 1-6, and 13 RP non-ATPases (Rpn), 1-3, 5-13, and 15. Similar to AAA-ATPases in prokaryotic ATP-dependent proteases, the Rpts form a hexameric ring that binds to the ends of the CP and is responsible for substrate unfolding and translocation into the CP. Unlike their prokaryotic counterparts, the Rpts are surrounded by non-ATPases. Apart from Rpn1, all Rpns form a cohesive struct...

We investigate the structure of mixed thin films composed of pentacene and diindenoperylene using x-ray reflectivity and grazing incidence x-ray diffraction. For equimolar mixtures we observe vanishing in-plane order coexisting with an excellent out-of-plane order, a yet unreported disordering behavior in binary mixtures of organic semiconductors, which are crystalline in their pure form. One approach to rationalize our findings is to introduce an anisotropic interaction parameter in the framework of a mean field model. By comparing the structural properties with those of other mixed systems, we discuss the effects of sterical compatibility and chemical composition on the mixing behavior, which adds to the general understanding of interactions in molecular mixtures.