Günter Pfeifer

A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the cop9-signalosome and eif3

The proteasome consists of a 20S proteolytic core particle (CP) and a 19S regulatory particle (RP), which selects ubiquitinated substrates for translocation into the CP. An eight-subunit subcomplex of the RP, the lid, can be dissociated from proteasomes prepared from a deletion mutant for Rpn10, an RP subunit. A second subcomplex, the base, contains all six proteasomal ATPases and links the RP to the CP. The base is sufficient to activate the CP for degradation of peptides or a nonubiquitinated protein, whereas the lid is required for ubiquitin-dependent degradation. By electron microscopy, the base and the lid correspond to the proximal and distal masses of the RP, respectively. The lid subunits share sequence motifs with components of the COP9/signalosome complex and eIF3, suggesting that these functionally diverse particles have a common evolutionary ancestry.

Near-atomic resolution structural model of the yeast 26S proteasome

The 26S proteasome operates at the executive end of the ubiquitin-proteasome 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.

Electron microscopy and image analysis of the multicatalytic proteinase

On electron micrographs, negatively stained multicatalytic proteinase molecules are viewed end‐on (ring shaped) or side‐on (rectangular shaped). For aurothioglucose, ammonium molybdate‐ and phosphotungstate‐stained molecules, the dimensions measured are consistent. In contrast, uranyl acetate‐staining reveals ring‐shaped particles which vary in diameter between 12 and 16 nm. This is due to a partial collapse and substantial flattening of the structure. Digital image analysis of side‐on views of the particles reveals a tripartite, reel‐shaped structure. Within the ring‐like, end‐on projections of ammonium molybdate‐stained molecules six local centres of mass can be discerned; their position appears to depart, however, from a true six‐fold symmetry.

Structure of the 26S proteasome with ATP-γS bound provides insights into the mechanism of nucleotide-dependent substrate translocation

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.

Deep Classification of a Large Cryo-Em Dataset Defines the Conformational Landscape of the 26S Proteasome.

Significance The 26S proteasome is a multisubunit molecular machine for the targeted degradation of intracellular proteins. It has an essential role in the maintenance of protein homeostasis. During its functional cycle the proteasome undergoes large-scale conformational changes. For a detailed mechanistic understanding, an analysis of its conformational landscape is indispensable. Capitalizing on a very large dataset of more than 3 million individual particles and using a novel image-classification strategy, we have been able to deconvolute coexisting conformational states. This led to the discovery of conformation intermediates that provide deeper insights into the sequence of events following the initial binding of ubiquitylated substrates. 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 (ATPh) and ATP-γS conformations, an intermediate state has been found. Its AAA-ATPase module adopts essentially the same topology that is observed in the ATPh 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.

The three-dimensional structure of proteasomes from Thermoplasma acidophilum as determined by electron microscopy using random conical tilting

The three‐dimensional structure of proteasomes from the archaebacterium Thermoplasma acidophilum has been determined to a resolution of approximately 2 nm from electron micrographs of negatively stained preparations using the method of random conical tilting. The particles turn out to be essentially cylinder‐shaped barrels, 15 nm long and 11 nm wide, enclosing a tripartite inner compartiment. An account is given of some of the present limitations which prevent to attain a higher resolution and possible ways to overcome these limitations are indicated.

Structure of the human 26S proteasome at a resolution of 3.9 Å

Significance The 26S proteasome is a giant protease assembled from at least 32 different canonical subunits. In eukaryotic cells it is responsible for the regulated degradation of proteins marked for destruction by polyubiquitin tags. Mainly because of the conformational heterogeneity of the 26S holocomplex, its structure determination has been challenging. Using cryo-electron microscopy single-particle analysis we were able to obtain a high-resolution structure of the human 26S proteasome allowing us to put forward an essentially complete atomic model. This model provides insights into the proteasome’s mechanism of operation and could serve as a basis for structure-based drug discovery. Protein degradation in eukaryotic cells is performed by the Ubiquitin-Proteasome 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.

The molecular chaperone TF55. Assessment of symmetry

TF55‐like factor from Sulfolobus solfataricus was purified to homogeneity and analyzed by electron microscopy and image analysis to determine the symmetries of these particles. Three different procedures were used to analyze the electron micrographs: (1) fuzzy‐set based classification of the particles according to their rotational power spectra; (2) multivariate statistical analysis based on singular value decomposition; (3) circular harmonic analysis. Averages obtained from the three methods show unequivocally that the TF55‐like complex presents a 9‐fold symmetry.

Structural characterization of the interaction of Ubp6 with the 26S proteasome

Significance In eukaryotic cells the 26S proteasome is responsible for the regulated degradation of intracellular proteins. The function of this large macromolecular machine is regulated by many cofactors, most notably the deubiquitylating enzyme ubiquitin C-terminal hydrolase 6 (Ubp6). Here, we investigate the structure of Ubp6 bound to the 26S proteasome and explore its influence on the conformational landscape of the 26S proteasome. Our structure reveals that Ubp6’s active site may contribute to a large composite active site, also formed by the mouth of the proteasomal ATPase ring and the active site of deubiquitylating enzyme regulatory particle non-ATPase 11. Moreover, Ubp6 modulates the conformational landscape of the proteasome, favoring an intermediate state, which may explain the effects of Ubp6 on proteasomal degradation. 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 intermediate-energy 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.

Electron microscopy and image analysis reveal common principles of organization in two large protein complexes: groEL-type proteins and proteasomes

In an attempt to settle the question of whether the multicatalytic proteinase or proteasome exist in all three kingdoms of life--eukaryotes, archaebacteria, and eubacteria--we have undertaken a search for them in the eubacterium Comamonas acidovorans. We have, in fact, isolated and purified a cylinder-shaped particle. However, according to various structural and biochemical criteria this turned out to be more reminiscent of the groEL protein from Escherichia coli and its homologs than to proteasomes of eukaryotic or archaebacterial origin. N-terminal sequencing provided definite proof for its belonging to this family of molecular chaperonins. Image analysis of electron micrographs revealed that the C. acidovorans groEL-like protein and proteasomes in spite of their significantly different dimensions have certain principles of organization in common.