Ubiquitin binding modulates the conformational dynamics and substrate degradation of the 26S proteasome

Abstract

The 26S proteasome is an ATP-dependent molecular machine that degrades ubiquitin-tagged proteins in several well-coordinated steps, including ubiquitin binding, substrate engagement, mechanical unfolding, de-ubiquitination, translocation, and proteolytic cleavage. Recently developed single-molecule FRET measurements, relying on the labeling of proteasome-incorporated unnatural amino acids, allow us to follow individual substrate proteins through the central channel and dissect the kinetics and coordination of processing steps. Mutational studies thereby reveal new details about the mechanisms of substrate engagement by the AAA+ ATPase motor and the contributions of individual ATPase subunits depending on their position in the hexameric spiral-staircase arrangement of the motor. Our FRET-based measurements with double-labeled proteasomes provide exciting insights into the conformational dynamics of the proteasomal regulatory particle and how ubiquitin-chain binding to certain receptor subunits influences the switching between substrate-free and substrate-processing states. Through this regulation of the proteasome conformational dynamics, ubiquitin chains accelerate substrate engagement by the AAA+ motor and potentiate mechanical unfolding, leading to faster and more efficient degradation. These effects depend on the length and linkage type of ubiquitin chains, indicating how the “ubiquitin code” may be utilized by the proteasome to prioritize substrates and fine-tune degradation in a complex cellular environment.