UC Berkeley

The 26S proteasome is responsible for selective protein degradation in eukaryotic cells. Polyubiquitin chains mark proteins for degradation by the proteasome, but before degradation can occur, ubiquitin chains must be removed by the intrinsic proteasomal deubiquitinase Rpn11. My graduate work primarily focused on understanding how Rpn11 is regulated so it only removes ubiquitin chains from substrates that have been committed to degradation. To answer this question, I developed an Rpn11 purification strategy and solved the crystal structure of Rpn11 alone and in complex with ubiquitin. These structures revealed several features that control the activity of Rpn11 toward ubiquitin substrates. I showed that Rpn11 is promiscuous in its activity toward different ubiquitin substrates, which explained how the enzyme is able to remove ubiquitin molecules from the wide range of proteasome substrate proteins. Comparison of our ubiquitin-bound and ubiquitin-free structures revealed that a critical loop changes conformation when ubiquitin binds to Rpn11 to form a catalytically active state and that substrate translocation may accelerate this transition, thereby activating the enzyme.

Rpn11 is part of a proteasome sub-complex called the lid. In the isolated lid Rpn11 is inhibited to reduce off pathway deubiquitination prior to proteasome assembly, but the mechanism of Rpn11 inhibition in the lid was not understood. We used cryo-EM to determine the atomic structure of the isolated lid which explained how Rpn11 is inhibited and how it becomes activated when incorporated into the proteasome. Our structure revealed that Rpn11 is held in a conformation that fully occludes its active site and prevents the enzyme from accessing ubiquitin substrates. Mutagenesis of inhibitory residues in the lid release Rpn11 from its inhibited state and activate the enzyme. Similarly, when the lid incorporates into the proteasome, Rpn11 is released from its inhibited state and adopts a fully extended, open conformation seen in the 26S proteasome.