Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach

TLDR

The 26S proteasome, central to the ubiquitin‑proteasome pathway, degrades intracellular proteins, yet while its 20S core particle structure is known, the 19S regulatory particle that recruits, unfolds, and translocates substrates remains structurally elusive. This study aims to delineate the molecular architecture of the 26S holocomplex using an integrative approach. The authors employed cryo‑electron microscopy, X‑ray crystallography, residue‑specific chemical cross‑linking, and proteomics to build the structure. The analysis revealed a modular lid composed of a horseshoe‑shaped Rpn3/5/6/7/9/12 heterohexamer that caps the AAA‑ATPase module, with Rpn8/Rpn11 positioned near the CP mouth, Rpn2 rigidly supporting the lid, Rpn1 variable at the ATPase periphery, and ubiquitin receptors Rpn10 and Rpn13 located distally, illustrating the sequential assembly of the proteasome.

Abstract

The 26S proteasome is at the executive end of the ubiquitin-proteasome pathway for the controlled degradation of intracellular proteins. While the structure of its 20S core particle (CP) has been determined by X-ray crystallography, the structure of the 19S regulatory particle (RP), which recruits substrates, unfolds them, and translocates them to the CP for degradation, has remained elusive. Here, we describe the molecular architecture of the 26S holocomplex determined by an integrative approach based on data from cryoelectron microscopy, X-ray crystallography, residue-specific chemical cross-linking, and several proteomics techniques. The “lid” of the RP (consisting of Rpn3/5/6/7/9/8/9/11/12) is organized in a modular fashion. Rpn3/5/6/7/9/12 form a horseshoe-shaped heterohexamer, which connects to the CP and roofs the AAA-ATPase module, positioning the Rpn8/Rpn11 heterodimer close to its mouth. Rpn2 is rigid, supporting the lid, while Rpn1 is conformationally variable, positioned at the periphery of the ATPase ring. The ubiquitin receptors Rpn10 and Rpn13 are located in the distal part of the RP, indicating that they were recruited to the complex late in its evolution. The modular structure of the 26S proteasome provides insights into the sequence of events prior to the degradation of ubiquitylated substrates.