Structure of the 26S proteasome with ATP-γS bound provides insights into the mechanism of nucleotide-dependent substrate translocation

Abstract

The 26S proteasome is a 2.5-MDa, ATP-dependent multisubunit proteolytic complex that processively destroys proteins carrying a degradation signal. The proteasomal ATPase heterohexamer is a key module of the 19S regulatory particle; it unfolds substrates and translocates them into the 20S core particle where degradation takes place. We used cryoelectron microscopy single-particle analysis to obtain insights into the structural changes of 26S proteasome upon the binding and hydrolysis of ATP. The ATPase ring adopts at least two distinct helical staircase conformations dependent on the nucleotide state. The transition from the conformation observed in the presence of ATP to the predominant conformation in the presence of ATP-γS induces a sliding motion of the ATPase ring over the 20S core particle ring leading to an alignment of the translocation channels of the ATPase and the core particle gate, a conformational state likely to facilitate substrate translocation. Two types of intersubunit modules formed by the large ATPase domain of one ATPase subunit and the small ATPase domain of its neighbor exist. They resemble the contacts observed in the crystal structures of ClpX and proteasome-activating nucleotidase, respectively. The ClpX-like contacts are positioned consecutively and give rise to helical shape in the hexamer, whereas the proteasome-activating nucleotidase-like contact is required to close the ring. Conformational switching between these forms allows adopting different helical conformations in different nucleotide states. We postulate that ATP hydrolysis by the regulatory particle ATPase (Rpt) 5 subunit initiates a cascade of conformational changes, leading to pulling of the substrate, which is primarily executed by Rpt1, Rpt2, and Rpt6.AAA ATPase | ubiquitin-proteasome pathway | hybrid methods in structural biology T he 26S proteasome is the executive arm of the ubiquitin-proteasome system (UPS), the major pathway for intracellular protein degradation in eukaryotic cells (1, 2). It degrades proteins that are marked for destruction by the covalent attachment of a polyubiquitin chain. The 2.5-MDa complex comprises a core particle (CP), the 20S proteasome, harboring the proteolytically active sites and one or two regulatory particles (RPs), which associate with the barrel-shaped CP. A key component of the RP is the AAA-ATPase module (ATPase associated with various cellular activities), which associates with the α-rings of the CP and prepares substrates for degradation. Whereas the 26S proteasome is the only soluble ATP-dependent protease in the eukaryotic cytosol, bacteria possess several different ATPdependent proteases like the ClpXP, HslUV, and the eubacterial proteasome-ARC/ Mpa (AAA ATPase forming ring-shaped complexes/mycobacterial proteasomal ATPase) system (3).The CP consists of four seven-membered rings (4). The two adjacent heteroheptameric β-rings form a cavity, which harbors the active sites (5, 6). The β-rings are sandwiched between two heteroheptameric α-rings, whose C-termini form a gate con...

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

“…This finding is consistent with the absence of extra density in s1-class reconstructions. Moreover, it is likely that the occupancy of a state in which the USP domain contacts Rpt1 also can be increased in the presence of ATP-γS or substrate, which induces switching to s3 (14,15). It has been proposed that the s1 state is primarily responsible for substrate recruitment, whereas both the s2 and s3 states are engaged in dealing with substrate (13).…”

“…A characteristic feature of the s2 state, as compared with the s1 state, is a better alignment of the channel axes of the CP and ATPase, suggesting that unfolded peptides can access the gated pore and catalytic cavity of the CP more easily. In a similar manner, the increased degradation of unfolded peptides by the 26S proteasome in the presence of ATP-γS (34) could be explained by a conformational change from the s1 state to the s3 state (14). In addition, binding of Ubp6 was reported to increase ATP hydrolysis (28).…”

“…Several cofactors interact transiently with this large macromolecular machine and modulate its function. The deubiquitylating enzyme ubiquitin C-terminal hydrolase 6 [Ubp6; ubiquitin-specific protease (USP) 14 in mammals] is the most abundant proteasome-interacting protein and has multiple roles in regulating proteasome function. Here, we investigate the structural basis of the interaction between Ubp6 and the 26S proteasome in the presence and absence of the inhibitor ubiquitin aldehyde.…”

“…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. A third conformation, s3, was found in the presence of the slowly hydrolysable ATP analogue ATP-γS (14) or upon the addition of polyubiquitylated substrate to 26S proteasomes with dysfunctional Rpn11 (15). Characteristics of the s3 state are a changed staircase arrangement of the AAA-module with Rpt1 most elevated and a further translation of the Rpns compared with s2, leaving Rpn11 and Rpn10 essentially invariant with respect to the mouth of the AAA-ATPase module.…”

Proteasomes, caught in the act

“…These conformational changes serve two critical functions during substrate degradation. First, they reposition the lid, base, and CP subcomplexes relative to one another, providing a clear path for the substrate through the complex [2,3]. Second, they cause the ATPases to grasp and pull downward on unstructured parts of the substrate via conserved, paddle-like loops that point into the pore of the ATPase ring ( Figure 1B).…”