The eukaryotic 26S proteasome utilizes a complex set of coordinated processing steps for the ATP‐dependent degradation of ubiquitin‐tagged substrates. Our cryo‐EM studies reveal important features of the proteasome regulatory particle, including a pronounced spiral‐staircase arrangement of its heterohexameric ATPase ring, that facilitate substrate engagement and degradation initiation. Substrate engagement induces a translocation‐competent conformation, in which the de‐ubiquitinating subunit Rpn11 is repositioned to function as a gatekeeper at the entrance of the processing pore and the ATPase ring adopts a distinct spiral‐staircase configuration, suggesting that highly coordinated ATP‐hydrolysis events drive substrate translocation. Optical tweezers single‐molecule studies of a related protease, ClpXP, further support such a coordinated ATP‐hydrolysis mechanism. Systematic mutational analyses of the proteasome ATPase ring, using a heterologous expression system and in‐vitro reconstitution of 26S holoenzymes, indicate that the six ATPase subunits play distinct roles in substrate engagement and translocation, corresponding to their positions in the spiral‐staircase arrangements of the ATPase ring. Furthermore, structural and mutational studies of Rpn11 provide new mechanistic insights into the translocation‐dependence and regulation of substrate de‐ubiquitination.

Energy-dependent proteases of the AAA+ family catalyze the highly specific protein degradation involved in cellular protein quality control and the regulation of numerous vital processes, yet the detailed mechanisms coupling ATP hydrolysis with mechanical substrate unfolding and translocation remain poorly understood. Our cryo-EM structural studies of the eukaryotic 26S proteasome show that its heterohexameric AAA+ ATPase ring adopts a conformation with pronounced spiral-staircase arrangement of subunits in the absence of substrate, but transitions into a translocation-competent conformation upon substrate engagement. This substrate-engaged ring conformation is characterized by uniform interfaces between the six ATPase subunit, a widened central channel coaxially aligned with the peptidase, and a rearranged, more planar spiral orientation of ATPase subunits that suggests a highly coordinated rapid progression of ATP-hydrolysis events around the ring. This coordinated ATP hydrolysis mechanism is further supported by our optical tweezers single-molecule studies of the related bacterial protease ClpXP and may be a general feature of AAA+ translocases. ClpXP translocates substrate polypeptides in steps with constant frequency but variable length, depending on the number of ATP-hydrolyzing subunits.

The 26S proteasome is the major protease in eukaryotic cells responsible for selective protein degradation to mediate protein quality control and regulation, yet the detailed mechanisms by which the proteasomal heterohexameric AAA+ unfoldase drives ATP‐dependent protein degradation remain poorly understood. Delineating the roles of the six distinct ATPase subunits in substrate processing has been hindered by limitations in working with endogenous proteasomes due to misassembly or lethal degradation defects. We therefore developed a heterologous expression system to produce the unfoldase subcomplex from Saccharomyces cerevisiae in Escherichia coli and reconstituted the proteasome in vitro to perform systematic mutational analyses of the individual ATPase subunits. Our studies demonstrate that the six ATPases have distinct functions in degradation, corresponding to their positions in the spiral staircases adopted by their large AAA+ domains in the absence or presence of substrate. ATP hydrolysis in subunits at the top of the staircases is critical for substrate engagement and translocation. Whereas the unfoldase relies on this vertical asymmetry for substrate processing, interaction with the peptidase exhibits a pronounced three‐fold symmetry. Only three ATPase subunits, arranged in alternate positions within the unfoldase ring, contain a conserved C‐terminal hydrophobic/aromatic/unspecified (HbYX) motif that is critical for both peptidase binding and gate‐opening, whereas the C‐terminal tails of the interjacent ATPase subunits are dispensable. Our study provides an initial glimpse into the potential importance of the spiral staircase configurations of proteasomal ATPase subunits in substrate processing and highlights how the 26S proteasome may deviate from simpler, homomeric AAA+ proteases.Grant Funding Source: Supported by US National Institutes of Health grant R01‐GM094497‐01A1

The 26S proteasome is the major eukaryotic ATP-dependent protease, yet the detailed mechanisms used by the proteasomal heterohexameric AAA+ unfoldase to drive substrate degradation remain poorly understood. To perform systematic mutational analyses of individual ATPase subunits, we heterologously expressed the unfoldase subcomplex from Saccharomyces cerevisiae in Escherichia coli and reconstituted the proteasome in vitro. Our studies demonstrate that the six ATPases have distinct roles in degradation, corresponding to their positions in the spiral staircases adopted by the AAA+ domains in the absence or presence of substrate. ATP hydrolysis in subunits at the top of the staircases is critical for substrate engagement and translocation. Whereas the unfoldase relies on this vertical asymmetry for substrate processing, interaction with the peptidase exhibits three-fold symmetry with contributions from alternate subunits. These diverse functional asymmetries highlight how the 26S proteasome deviates from simpler, homomeric AAA+ proteases.

The 26S proteasome is the major ATP-dependent protease in eukaryotes and thus involved in regulating a diverse array of vital cellular processes. Three subcomplexes form this massive degradation machine: the lid, the base, and the core. While assembly of base and core has been well-studied, the detailed molecular mechanisms involved in formation of the nine-subunit lid remain largely unknown. Here, we reveal that helices found at the C terminus of each lid subunit form a helical bundle that directs the ordered self-assembly of the lid subcomplex. Furthermore, we use an integrative modeling approach to gain critical insights into the bundle topology and provide an important structural framework for our biochemical data. We show that the helical bundle serves as a hub through which the last-added subunit Rpn12 monitors proper lid assembly before incorporation into the proteasome. Finally, we predict that the assembly of the COP9 signalosome depends on a similar helical bundle.

The proteasome is the major ATP-dependent protease in eukaryotic cells, but limited structural information restricts a mechanistic understanding of its activities. The proteasome regulatory particle, consisting of the lid and base subcomplexes, recognizes and processes polyubiquitinated substrates. Here we used electron microscopy and a new heterologous expression system for the lid to delineate the complete subunit architecture of the regulatory particle from yeast. Our studies reveal the spatial arrangement of ubiquitin receptors, deubiquitinating enzymes and the protein unfolding machinery at subnanometre 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.

In eukaryotic cells, the proteasome degrades unwanted proteins by recognizing specific polyubiquitin tags covalently attached to these proteins. The precise manner in which these ubiquitin chains are recognized and removed from the targeted proteins prior to proteolysis is poorly understood. This is partly due to a lack of structural information on the ubiquitin-recognizing components of the proteasome 19S regulatory particle. Using a recombinant expression system and electron microscopy, we were able to localize all subunits of the yeast 19S particle, and elucidate the spatial arrangement of ubiquitin receptors, deubiquitinating enzymes, and the protein unfolding machinery. Our studies also revealed large conformational rearrangements in the lid subcomplex upon holoenzyme formation, suggesting an allosteric mechanism for activation of its deubiquitination activity. From these studies, we have a much better understanding of the manner in which the 26S recognizes and deubiquitinates proteins marked for proteolysis.

For example, we can utilize this X-ray resultant force for the trapping of nanoprobe and the crystal growth azimuth control during crystal growths. In addition, we proved that the ultra-fast DXT using protein molecules labeled the gold nanocrystal [2] can detect aN level’s force field in functional protein molecules. In the future, we can detect dynamic structural changes of functional surface induced by an ultra-small force field that cannot be detected by STM and AFM. [1] Y. C. Sasaki et al., Appl. Phys. Lett., 89, 053121(2006). [2] H. Shimizu et al., Cell, 132, 67–78 (2008).