An AAA Motor-Driven Mechanical Switch in Rpn11 Controls Deubiquitination at the 26S Proteasome
Evan J Worden1, Ken C Dong2, Andreas Martin2
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA.
Abstract:
Poly-ubiquitin chains direct protein substrates to the 26S proteasome, where they are removed by the deubiquitinase Rpn11 during ATP-dependent substrate degradation. Rapid deubiquitination is required for efficient degradation but must be restricted to committed substrates that are engaged with the ATPase motor to prevent premature ubiquitin chain removal and substrate escape. Here we reveal the ubiquitin-bound structure of Rpn11 from S. cerevisiae and the mechanisms for mechanochemical coupling of substrate degradation and deubiquitination. Ubiquitin binding induces a conformational switch of Rpn11's Insert-1 loop from an inactive closed state to an active β hairpin. This switch is rate-limiting for deubiquitination and strongly accelerated by mechanical substrate translocation into the AAA+ motor. Deubiquitination by Rpn11 and ubiquitin unfolding by the ATPases are in direct competition. The AAA+ motor-driven acceleration of Rpn11 is therefore important to ensure that poly-ubiquitin chains are removed only from committed substrates and fast enough to prevent their co-degradation.
Insights
The deubiquitinase Rpn11
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- Poly-ubiquitin chains target proteins for degradation by the 26S proteasome.
- The deubiquitinase Rpn11 removes ubiquitin chains during proteasomal degradation.
- Efficient degradation requires rapid deubiquitination, but only for committed substrates.
Purpose of the Study:
- To elucidate the structure of ubiquitin-bound Rpn11 from S. cerevisiae.
- To understand the mechanisms coupling substrate degradation and deubiquitination.
- To investigate how Rpn11 activity is regulated during proteasomal processing.
Main Methods:
- X-ray crystallography to determine the ubiquitin-bound structure of Rpn11.
- Biochemical assays to study deubiquitination kinetics.
- Mechanistic studies on the interaction between Rpn11 and the AAA+ ATPase motor.
Main Results:
- The ubiquitin-bound structure of Rpn11 reveals a conformational switch in its Insert-1 loop.
- Ubiquitin binding induces an inactive closed state to an active beta hairpin.
- Mechanical translocation of substrate by the AAA+ motor accelerates Rpn11 activity, which is rate-limiting for deubiquitination.
Conclusions:
- Rpn11's conformational switch is crucial for regulated deubiquitination.
- Mechanical force from the AAA+ motor accelerates Rpn11 activity.
- This ensures ubiquitin removal only from committed substrates, preventing premature release and co-degradation.
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