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.
Ubiquitin-specific protease 7 (USP7) deubiquitinase activity is controlled by a number of regulatory factors, including stimulation by intramolecular accessory domains. Alone, the USP7 catalytic domain (USP7cd) shows limited activity and apo USP7cd crystal structures reveal a disrupted catalytic triad. By contrast, ubiquitin-conjugated USP7cd structures demonstrate the canonical cysteine protease active-site geometry; however, the structural features of the USP7cd that stabilize the inactive conformation and the mechanism of transition between inactive and active states remain unclear. Here we use comparative structural analyses, molecular dynamics simulations, and in silico sequence re-engineering via directed sampling by RosettaDesign to identify key molecular determinants of USP7cd activation and successfully engineer USP7cd for improved activity. Full kinetic analysis and multiple X-ray crystal structures of our designs indicate that electrostatic interactions in the distal "switching loop" region and local packing in the hydrophobic core mediate subtle but significant conformational changes that modulate USP7cd activation.
Ubiquitin specific protease 7 (USP7) regulates the protein stability of key cellular regulators in pathways ranging from apoptosis to neuronal development, making it a promising therapeutic target. Here we used an engineered, activated variant of the USP7 catalytic domain to perform structure−activity studies of electrophilic peptidomimetic inhibitors. Employing this USP7 variant, we found that inhibitors with a cyanopyrrolidine warhead unexpectedly promoted a β-elimination reaction of the initial covalent adducts, thereby converting the active-site cysteine residue to dehydroalanine. We determined that this phenomenon is specific for the USP7 catalytic cysteine and that structural features of the inhibitor and protein microenvironment impact elimination rates. Using comprehensive docking studies, we propose that the characteristic conformational dynamics of USP7 allow access to conformations that promote the ligand-induced elimination. Unlike in conventional reversible-covalent inhibition, the compounds described here irreversibly destroy a catalytic residue while simultaneously converting the inhibitor to a nonelectrophilic byproduct. Accordingly, this unexpected finding expands the scope of covalent inhibitor modalities and offers intriguing insights into enzyme−inhibitor dynamics.
A cellular membrane affinity chromatography (CMAC) column has been created using cellular membrane fragments from a 1321N1 cell line stably transfected with the P2Y1 receptor. The CMAC(1321N1P2Y1) column contained functional P2Y1 and histamine 1 receptors, which independently bound receptor-specific ligands. The data obtained with the CMAC(1321N1P2Y1) column demonstrate that multiple-G-protein coupled receptor (GPCR) columns can be developed and used to probe interactions with the immobilized receptors and that endogenously expressed GPCRs can be used to create CMAC columns. The results also establish that the histamine 1 receptor can be immobilized with retention of ligand-specific binding.
15The 26S proteasome is the principle 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 20 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 25 kinetic proofreading mechanism to ensure degradation fidelity and substrate prioritization.
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