Crystal structure of the proteasomal deubiquitylation module Rpn8-Rpn11

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• The 26S proteasome facilitates ATP-dependent protein degradation, crucial for cellular homeostasis.
• Rpn11, a proteasome subunit, possesses isopeptidase activity essential for removing polyubiquitin chains from substrates.
• Understanding Rpn11's regulation is key to comprehending proteasomal degradation pathways.

Purpose of the Study:

• To elucidate the structural basis of Rpn11's deubiquitylation activity.
• To investigate the mechanisms regulating Rpn11 activation within the 26S proteasome.
• To provide insights into how Rpn11's activity is coupled to proteasomal substrate processing.

Main Methods:

• X-ray crystallography was used to determine the structures of Rpn8-Rpn11 fusion protein complexes.
• Cryo-EM data was utilized to dock the Rpn11 structure into the 26S proteasome.
• Biochemical assays assessed the deubiquitylation activity of the Rpn11 fusion protein.

Main Results:

• Three crystal structures of the Rpn8-Rpn11 heterodimer in complex with a nanobody were obtained.
• The Rpn8-Rpn11 fusion protein showed modest deubiquitylation activity, which is enhanced upon incorporation into the 26S proteasome.
• Structural analysis revealed features preventing premature activation, including low ubiquitin affinity and a blocking insertion segment.

Conclusions:

• Rpn11 activation is dependent on its integration into the 26S proteasome and ATP hydrolysis.
• Structural elements like an insertion segment and catalytic loop conformation regulate Rpn11 activity.
• Interactions with proteasomal ATPase subunits stabilize Rpn11's active conformation and enhance ubiquitin binding, preventing off-target deubiquitylation.