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

Abstract

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...

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

“…1C), indicating less structural heterogeneity. The overall structure differs notably from the reconstruction from 26S proteasomes alone, which predominantly adopt the s1 conformation (6), and appears more similar to the s2 conformation (13). Most importantly, two extra densities in the direct vicinity of Rpn1 are clearly distinguishable.…”

“…Previous analysis revealed that the conformational states of the RPs at the two CP ends are not correlated (6,13,15). Therefore, the following analysis was performed using the holocomplex cut into two halves (6, 13), i.e., the two RPs of the double-capped 26S proteasomes were treated as separate particles.…”

“…In ATP-containing buffer, purified 26S proteasomes primarily adopt the s1 state, which is characterized by pronounced off-axis positioning of the AAAATPase hexamer with respect to the CP and a staircase arrangement of the Rpts with Rpt3 in the most elevated position (6)(7)(8)(9). Under the same conditions a minority of particles (∼20%) adopts the s2 state, in which the axis of the AAA-ATPase is positioned closer to that of the CP and the Rpns concomitantly rotate largely en bloc by ∼25° (13). As a consequence of this Rpn motion, the active site of Rpn11 becomes accessible to the polyubiquitin chain of the substrate, and the ubiquitin receptor Rpn10 is positioned closer to the AAA-ATPase module.…”

“…At least three distinct states, which we refer to as "s1-s3," can be distinguished (13). In ATP-containing buffer, purified 26S proteasomes primarily adopt the s1 state, which is characterized by pronounced off-axis positioning of the AAAATPase hexamer with respect to the CP and a staircase arrangement of the Rpts with Rpt3 in the most elevated position (6)(7)(8)(9).…”

Conformational Preference and Spectroscopical Characteristics of the Active Pharmaceutical Ingredient Levetiracetam

“…[26][27][28][29][30][31][32][33][34][35] This kind of information is also crucial for modeling the drug-receptor interactions 36 or for understanding the thermodynamics and kinetics of crystal growth processes 37,38 and, consequently, the polymorphism associated with pharmaceutical compounds. 25 Yet another reason for the conformational landscape analysis of a drug molecule is that it can suffer large structural changes during its functional cycle, 39 so that the conformation of the bioactive conformer could be significantly different from those of the most stable one in a particular solvent or in gas phase. 40 The conformational landscape of LEV in water and dichloromethane was studied previously by Li and Si 40 who identified 9 conformers.…”