Structure of the 26S proteasome with ATP-γS bound provides insights into the mechanism of nucleotide-dependent substrate translocation - PubMed (original) (raw)

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

Paweł Śledź et al. Proc Natl Acad Sci U S A. 2013.

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Comparison of the ATP-γS–bound structure and ATPh structure. (Upper) Pseudoatomic ATP-γS model fitted into the cryo-EM reconstruction of the 26S proteasome (gray) in three different views. (Lower) Corresponding views of ATPh model (PDB ID code: 4B4T). The dashed lines mark the positions of Rpn13 (Left and Center) and Rpn6 (Right) to facilitate the comparison between Upper and Lower.

Fig. 2.

Fig. 2.

Intersubunit modules in the AAA ring. (A and B) Structures of the AAA-ATPase for the ATP-γS structure and the ATPh structure, respectively. The subunits Rpt1, Rpt6, and Rpt4 are shown in blue and Rpt2, Rpt3, and Rpt5 in cyan. Large and small AAA domains of each subunit are shown in darker and lighter shades, respectively. ISMs are annotated by black (closed conformation) and red (open) borders. (C and D) Structure of ISM1–5 in its closed and open conformations, respectively. Rotation of the small domain by ∼21° is required for the conformational transition between the two states. (E) Nucleotide-binding sites of Rpt5 and Rpt4 in the ATP-γS structure. For the closed ISM (ISM4–5) the Arg finger (magenta triangle) is placed in the proximity of nucleotide-binding site of Rpt4 (shown in red), allowing for interaction. For the open conformation (ISM5–1), the Arg finger rotates away from the nucleotide-binding site of Rpt5 (white); this rearrangement prevents an engagement of the Arg finger in binding. (F and G) The two lockwasher-like topologies of the AAA-ATPase hexamer. Continuity of interactions between the neighboring subunits is illustrated by the involvement of the Arg finger in nucleotide binding at the adjacent large domain. The introduction of the open ISM (enclosed in red) allows closure of the ring.

Fig. 3.

Fig. 3.

Displacement of small AAA domains of Rpt subunits with respect to the two adjacent α-subunits upon the change of the nucleotide state. Arrangements in the ATPh conformation are shown in color and the ATP-γS arrangements in gray.

Fig. 4.

Fig. 4.

Alignment of the AAA-ATPase translocation channels and the α-ring gate. (A and B) The AAA ring and the α ring in ATP-γS–bound (A) and ATPh conformation (B). (C and D) OB ring and α ring. (E and F) Schematic of channels of α, AAA, and OB ring for ATP-γS (E) and ATP (F) conformations.

Fig. 5.

Fig. 5.

Effects of transition from γS- (Left) to ATPh conformation on substrate translocation. (Left) Differences of vertical position of pore loops that are thought to pull the substrate. (Center and Right) Cut-through views through ATP-γS and ATPh structures show how alignment of the rings affects the substrate path. The pore loops of Rpt1 and Rpt3 are marked with stars.

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