The activated ClpP peptidase forcefully grips a protein substrate.
Steven D Walker1, Adrian O Olivares2
1Department of Biochemistry, Vanderbilt University, Nashville, Tennessee; Chemical and Physical Biology Graduate Program, Vanderbilt University, Nashville, Tennessee.
Biophysical Journal
|September 1, 2022
Summary
The caseinolytic peptidase P (ClpP) grips protein substrates with significant force, revealing its mechanical role in protein degradation. This peptidase activity is not required for substrate binding, offering new insights into AAA+ protease mechanisms.
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- ATPases associated with diverse cellular activities (AAA+) proteases maintain protein homeostasis by coupling ATP hydrolysis to protein unfolding and degradation.
- The mechanical contribution of the peptidase component to substrate denaturation and threading remains poorly understood.
Purpose of the Study:
- To investigate the mechanical activity of the caseinolytic peptidase P (ClpP) from Escherichia coli.
- To elucidate the role of ClpP in substrate binding and mechanical unfolding, independent of partner ATPases.
Main Methods:
- Single-molecule optical trapping was employed to measure mechanical forces and bond lifetimes between ClpP and protein substrates.
- Experiments were conducted with and without an activating small-molecule acyldepsipeptide.
Main Results:
- ClpP engages protein substrates with mechanical loads exceeding 40 pN, surpassing forces observed for AAA+ unfoldases and protease complexes.
- Substrate-ClpP bond rupture forces exhibited slip bond behavior, independent of ClpP's peptidase activity.
- Unloaded bond lifetimes reached up to ~160 seconds for difficult-to-unfold proteins, relevant to protein unfolding timescales.
Conclusions:
- ClpP possesses significant mechanical gripping capabilities crucial for AAA+ protease function.
- The mechanical interaction of ClpP with substrates is distinct from its enzymatic peptidase activity.
- These findings provide direct mechanical insights into how AAA+ proteases unfold and degrade protein substrates.
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