The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together

Abstract

Proteasomes execute the degradation of most cellular proteins. Although the 20S core particle (CP) has been studied in great detail, the structure of the 19S regulatory particle (RP), which prepares ubiquitylated substrates for degradation, has remained elusive. Here, we report the crystal structure of one of the RP subunits, Rpn6, and we describe its integration into the cryo-EM density map of the 26S holocomplex at 9.1 Å resolution. Rpn6 consists of an α-solenoid-like fold and a proteasome COP9/signalosome eIF3 (PCI) module in a right-handed suprahelical configuration. Highly conserved surface areas of Rpn6 interact with the conserved surfaces of the Pre8 (alpha2) and Rpt6 subunits from the alpha and ATPase rings, respectively. The structure suggests that Rpn6 has a pivotal role in stabilizing the otherwise weak interaction between the CP and the RP.

Fig. 1.

Crystal structure of Rpn6. (A) Domain structure of Rpn6. The purple region denotes a capping helix; the yellow region is predicted to be α-helical. (B) Ribbon representation of Rpn6, colored by domain structure. Two views related by 90° rotation are shown. N and C termini and selected secondary structure elements are indicated. (C) Detailed view of the interface between the solenoid fold and the PCI module.

Fig. 2.

Surface analysis of Rpn6. (A) Surface conservation mapped onto the surface of Rpn6. On the left, the same orientation as in Fig. 1A is shown. The similarity score from a multiple alignment of 21 related sequences (Fig. S2) was mapped onto the molecular surface of Rpn6. A cyan-white-magenta color gradient indicates increasing surface conservation. Regions I, II, and III are indicated. (B) Side views on the winged-helix subdomain. (C) The predicted C-terminal helix. The helix is represented as a helical wheel, and residue properties are indicated. (Left) Conservation is represented using the same color scheme as in panel A. (Right) Hydrophobic side chains are indicated in yellow. Positively and negatively charged functional groups are colored blue and red, respectively. The rest of the surface is shown in white.

Fig. 3.

Binary interaction of Rpn6 and Rpn7. (A) Probing for direct interactions of Rpn6 with lid particle subunits. Purified Rpn6 was incubated individually with His-tagged Rpn5, Rpn7, Rpn8, or Rpn9 from D. melanogaster. The Coomassie-stained SDS-PAGE gels show the initial mixtures, unbound proteins, and proteins precipitated with Ni-affinity resin. (B) Location of Rpn6 mutations in the structure. The respective amino acid residue substitutions are indicated. Mutated residues are shown in space-filling mode. (C) Excerpts from Rpn6 sequence alignment showing the mutated regions. (D) Both the PCI module interface region III and the C-terminal helix of Rpn6 are required for the interaction with Rpn7. His-tagged Rpn7 was incubated with either wild-type Rpn6(30–422) or Rpn6(30–422) mutants M1, M2, M3, or M4 and analyzed as described for panel A.

Fig. 4.

Location of Rpn6 in the 26S proteasome. (A) Rpn6 density within the 9 Å cryo-EM density of the 26S proteasome from S. pombe. Three views are shown. The lid, base, and core subcomplex densities are indicated in gold, blue, and red, respectively. Density ascribed to Rpn6 is colored green. The core particle is clipped off at the β₇ ring. (B) Detailed view of the Rpn6 fitted into the EM envelope. Density assigned for Rpn6 was segmented from the map. The homology model of Rpn6 from S. pombe including the predicted N-terminal helices α(-1) and α0 are included. Similar orientations as in panel A are shown.

Fig. 5.

Putative interactions of Rpn6 with Pre8 and Rpt6. (A) Excerpts from Pre8 and Rpt6 sequence alignments for the Rpn6 contact regions. (B and C) Detailed view of the interactions with Pre8. (Upper) The similarity score of an extensive alignment of Pre8 sequences mapped onto the homology model surface. Rpn6 is shown as a green ribbon. (Lower) Putative key interactions at the interface. Both proteins are shown in ribbon representation. Selected side chains are shown as sticks. Putative hydrogen bonds are indicated by dashed lines. (D and E) Detailed view of the interactions with Rpt6.