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Structural insights into ClpP protease side exit pore-opening by a pH drop coupled with substrate hydrolysis
Leehyeon Kim1, Byung-Gil Lee1, Minki Kim1
1Department of Life Sciences, Korea University, Seoul, South Korea.
The EMBO Journal
|May 20, 2022
Summary
Acyclic depsipeptides (ADEPs) activate the ClpP protease, a key bacterial machine. New structures reveal how ClpP releases peptide products, offering insights into antibiotic development.
Area of Science:
- Biochemistry
- Structural Biology
- Microbiology
Background:
- ClpP serine peptidase is a tetradecameric machine crucial for cellular processes.
- Acyclic depsipeptides (ADEPs) activate ClpP without ATPases, showing antibiotic potential.
- Previous studies detailed ClpP structure and activation, but product release mechanisms remain unclear.
Purpose of the Study:
- To elucidate the molecular mechanism of product release from the ClpP protease.
- To investigate the conformational changes and driving forces behind product release.
- To understand the role of pH in ClpP-mediated hydrolysis and product egress.
Main Methods:
- X-ray crystallography of Bacillus subtilis ClpP (BsClpP) bound to ADEPs.
- Cryo-electron microscopy (Cryo-EM) to capture various BsClpP conformational states.
- Enzymatic assays measuring pH changes during substrate hydrolysis by BsClpP and proteinase K.
Main Results:
- Novel crystal structures of BsClpP in ADEP-bound states were determined.
- Cryo-EM revealed diverse BsClpP conformations influenced by pH.
- Substrate hydrolysis by BsClpP and proteinase K demonstrably lowered pH.
- A link between substrate hydrolysis, pH reduction, and product release was established.
Conclusions:
- The study provides structural and mechanistic insights into ADEP-activated ClpP.
- Product release from ClpP is facilitated by a pH-lowering mechanism driven by hydrolysis.
- Findings advance understanding of ClpP self-compartmentalizing protease function and antibiotic development.
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