The proteasome is the major ATP-dependent protease in eukaryotic cells, but limited structural information strongly restricts a mechanistic understanding of its activities. The proteasome regulatory particle, consisting of the lid and base subcomplexes, recognizes and processes poly-ubiquitinated substrates. We used electron microscopy and a newly-developed heterologous expression system for the lid to delineate the complete subunit architecture of the regulatory particle. Our studies reveal the spatial arrangement of ubiquitin receptors, deubiquitinating enzymes, and the protein unfolding machinery at subnanometer resolution, outlining the substrate’s path to degradation. Unexpectedly, the ATPase subunits within the base unfoldase are arranged in a spiral staircase, providing insight into potential mechanisms for substrate translocation through the central pore. Large conformational rearrangements of the lid upon holoenzyme formation suggest allosteric regulation of deubiquitination. We provide a structural basis for the ability of the proteasome to degrade a diverse set of substrates and thus regulate vital cellular processes.

Graphical AbstractHighlights d The rate-limiting step of protein degradation by the proteasome is unfolding d Substrate contact with the motor triggers a conformational switch of the proteasome d Substrates with poor initiation regions are quickly rejected d Supernumerary ubiquitin chains promote degradation of otherwise poor substrates A complete kinetic picture of proteasomal degradation reveals that the engagement steps prior to substrate commitment are fast relative to subsequent deubiquitination, translocation, and unfolding. SUMMARYThe 26S proteasome is the principal macromolecular machine responsible for protein degradation in eukaryotes. However, little is known about the detailed kinetics and coordination of the underlying substrate-processing steps of the proteasome, and their correlation with observed conformational states.Here, we used reconstituted 26S proteasomes with unnatural amino-acid-attached fluorophores in a series of FRET-and anisotropy-based assays to probe substrate-proteasome interactions, the individual steps of the processing pathway, and the conformational state of the proteasome itself. We develop a complete kinetic picture of proteasomal degradation, which reveals that the engagement steps prior to substrate commitment are fast relative to subsequent deubiquitination, translocation, and unfolding. Furthermore, we find that non-ideal substrates are rapidly rejected by the proteasome, which thus employs a kinetic proofreading mechanism to ensure degradation fidelity and substrate prioritization.(A) Schematic for the substrate processing pathway. Ubiquitin chains (pink) target a substrate (gold) to the 26S proteasome in the s1 state. The substrate's unstructured tail is inserted into the pore of the AAA + motor (blue), where it interacts with pore loops (red) that drive translocation. After substrate engagement, the regulatory particle changes its conformation to an s3-like state and Rpn11 (green) shifts to a central position that allows translocation-coupled deubiquitination. Ubiquitin-chain removal is followed by mechanical substrate unfolding and threading of the polypeptide into the 20S core for proteolytic cleavage.

SUMMARY Substrates are targeted for proteasomal degradation through the attachment of ubiquitin chains that need to be removed by proteasomal deubiquitinases prior to substrate processing. In budding yeast, the deubiquitinase Ubp6 trims ubiquitin chains and affects substrate processing by the proteasome, but the underlying mechanisms and its location within the holoenzyme remained elusive. Here we show that Ubp6 activity strongly responds to interactions with the base ATPase and the conformational state of the proteasome. Electron-microscopy analyses reveal that ubiquitin-bound Ubp6 contacts the N-ring and AAA+ ring of the ATPase hexamer, in close proximity to the deubiquitinase Rpn11. Ubiquitin-bound Ubp6 inhibits substrate deubiquitination by Rpn11, stabilizes the substrate-engaged conformation of the proteasome, and allosterically interferes with the engagement of a subsequent substrate. Ubp6 may thus act as an ubiquitin-dependent timer to coordinate individual processing steps at the proteasome and modulate substrate degradation.

Engineered destruction of target proteins by recruitment to the cell’s degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.