Structural Models for Interactions between the 20S Proteasome and Its PAN/19S Activators (original) (raw)
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Structure, 2009
Intrinsic conformational transitions contribute to the catalytic action of many enzymes. Here we use a single-molecule approach to demonstrate how such transitions are linked to the catalytic sites of the eukaryotic proteasome, an essential protease of the ubiquitin pathway. The active sites of the cylindrical proteasomal core particle (CP) are located in a central chamber accessible through gated entry channels. By atomic force microscopy (AFM), we found continual alternation between open and closed gate conformations. We analyzed the relative abundance of these conformers in wild-type and mutated yeast CPs upon exposure to substrates or inhibitors. Our data indicate that the dynamic gate can be opened by allosteric coupling to a tetrahedral transition state at any of the working active centers. The results point to the N α -amine of the N-terminal active site threonyl residue as the major effector group responsible for triggering the essential conformational switch.
Mechanism of Gate Opening in the 20S Proteasome by the Proteasomal ATPases
Molecular Cell, 2008
Substrates enter the cylindrical 20S proteasome through a gated channel that is regulated by the ATPases in the 19S regulatory particle in eukaryotes or the homologous PAN ATPase complex in archaea. These ATPases contain a conserved C-terminal hydrophobic-tyrosine-X (HbYX) motif that triggers gate opening upon ATP binding. Using cryo-electron microscopy, we identified the sites in the archaeal 20S where PAN's C-terminal residues bind and determined the structures of the gate in its closed and open forms. Peptides containing the HbYX motif bind to 20S in the pockets between neighboring a subunits where they interact with conserved residues required for gate opening. This interaction induces a rotation in the a subunits and displacement of a reverse-turn loop that stabilizes the open-gate conformation. This mechanism differs from that of PA26/28, which lacks the HbYX motif and does not cause a subunit rotation. These findings demonstrated how the ATPases' C termini function to facilitate substrate entry.
Journal of Biological Chemistry, 2008
The 26 S proteasome is an energy-dependent protease that degrades proteins modified with polyubiquitin chains. It is assembled from two multi-protein subcomplexes: a protease (20 S proteasome) and an ATPase regulatory complex (PA700 or 19 S regulatory particle) that contains six different AAA family subunits (Rpt1 to -6). Here we show that binding of PA700 to the 20 S proteasome is mediated by the COOH termini of two (Rpt2 and Rpt5) of the six Rpt subunits that constitute the interaction surface between the subcomplexes. COOH-terminal peptides of either Rpt2 or Rpt5 bind to the 20 S proteasome and activate hydrolysis of short peptide substrates. Simultaneous binding of both COOH-terminal peptides had additive effects on peptide substrate hydrolysis, suggesting that they bind to distinct sites on the proteasome. In contrast, only the Rpt5 peptide activated hydrolysis of protein substrates. Nevertheless, the COOH-terminal peptide of Rpt2 greatly enhanced this effect, suggesting that proteasome activation is a multistate process. Rpt2 and Rpt5 COOH-terminal peptides cross-linked to different but specific subunits of the 20 S proteasome. These results reveal critical roles of COOH termini of Rpt subunits of PA700 in the assembly and activation of eukaryotic 26 S proteasome. Moreover, they support a model in which Rpt subunits bind to dedicated sites on the proteasome and play specific, nonequivalent roles in the asymmetric assembly and activation of the 26 S proteasome.
Insights into the molecular architecture of the 26S proteasome
Proceedings of the National Academy of Sciences, 2009
Cryo-electron microscopy in conjunction with advanced image analysis was used to analyze the structure of the 26S proteasome and to elucidate its variable features. We have been able to outline the boundaries of the ATPase module in the ''base'' part of the regulatory complex that can vary in its position and orientation relative to the 20S core particle. This variation is consistent with the ''wobbling'' model that was previously proposed to explain the role of the regulatory complex in opening the gate in the ␣-rings of the core particle. In addition, a variable mass near the mouth of the ATPase ring has been identified as Rpn10, a multiubiquitin receptor, by correlating the electron microscopy data with quantitative mass spectrometry.
Proceedings of the National Academy of Sciences, 1999
We present a biochemical and crystallographic characterization of active site mutants of the yeast 20S proteasome with the aim to characterize substrate cleavage specificity, subunit intermediate processing, and maturation. 1(Pre3), 2(Pup1), and 5(Pre2) are responsible for the postacidic, tryptic, and chymotryptic activity, respectively. The maturation of active subunits is independent of the presence of other active subunits and occurs by intrasubunit autolysis. The propeptides of 6(Pre7) and 7(Pre4) are intermediately processed to their final forms by 2(Pup1) in the wild-type enzyme and by 5(Pre2) and 1(Pre3) in the 2(Pup1) inactive mutants. A role of the propeptide of 1(Pre3) is to prevent acetylation and thereby inactivation. A gallery of proteasome mutants that contain active site residues in the context of the inactive subunits 3(Pup3), 6(Pre7), and 7(Pre4) show that the presence of Gly-1, Thr1, Asp17, Lys33, Ser129, Asp166, and Ser169 is not sufficient to generate activity.
Structure and structure formation of the 20S proteasome
Molecular biology reports, 1997
Eukaryotic 20S proteasomes are complex oligomeric proteins. The maturation process of the 14 different alpha- and beta-subunits has to occur in a highly coordinate manner. In addition beta-subunits are synthesized as proproteins and correct processing has to be guaranteed during complex maturation. The structure formation can be subdivided in different phases. The knowledge of the individual phases is summarized in this publication. As a first step the newly synthesized monomers have to adopt the correct tertiary structure, a process that might be supported in the case of the beta-subunits by the intramolecular chaperone activity postulated for the prosequences. Subsequently the alpha-subunits form ring-like structures thereby providing docking sites for the different beta-subunits. The result most likely is a double ring structure (13S precursor) representing half-proteasomes, which contain immature proproteins. Two 13S precursors associate to form the proteolytically inactive 16S ...
The central unit within the 19S regulatory particle of the proteasome
Nature Structural & Molecular Biology, 2008
The 26S proteasome is a multisubunit enzyme composed of a cylindrical catalytic core (20S) and a regulatory particle (19S) that together perform the essential degradation of cellular proteins tagged by ubiquitin. To date, however, substrate trajectory within the complex remains elusive. Here we describe a previously unknown functional unit within the 19S, comprising two subunits, Rpn1 and Rpn2. These toroids physically link the site of substrate recruitment with the site of proteolysis. Rpn2 interfaces with the 20S, whereas Rpn1 sits atop Rpn2, serving as a docking site for a substrate-recruitment factor. The 19S ATPases encircle the Rpn1-Rpn2 stack, covering the remainder of the 20S surface. Both Rpn1-Rpn2 and the ATPases are required for substrate translocation and gating of the proteolytic channel. Similar pairing of units is found in unfoldases and nuclear transporters, exposing common features of these protein nanomachines.
The Axial Channel of the 20S Proteasome Opens Upon Binding of the PA200 Activator
Journal of Molecular Biology, 2005
Proteasomes consist of a proteolytic core called the 20 S particle and ancillary factors that regulate its activity in various ways. PA200 has been identified as a large (200 kDa) nuclear protein that stimulates proteasomal hydrolysis of peptides. To characterize its interaction with the 20 S core, we have visualized PA200-20 S complexes by electron microscopy. Monomers of PA200 bind to one or both ends of the 20 S core. Reconstructed in three dimensions to 23 Å resolution from cryo-electron micrographs of the singly bound complex, PA200 has an asymmetric dome-like structure with major and minor lobes. Taking into account previous bioinformatic analysis, it is likely to represent an irregular folding of an a-helical solenoid composed of HEAT-like repeats. PA200 makes contact with all a-subunits except a7, and this interaction induces an opening of the axial channel through the a-ring. Thus, the activation mechanism of PA200 is expressed via its allosteric effects on the 20 S core particle, perhaps facilitating release of digestion products or the entrance of substrates.