Escherichia coli ClpB is a non-processive polypeptide translocase

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Escherichia coli caseinolytic protease (Clp)B is a hexameric AAA+ enzyme vital for proteome maintenance.
• ClpB's ability to catalyze protein disaggregation is essential for cellular health.
• Previous studies suggested ClpB translocates polypeptides through its axial channel.

Purpose of the Study:

• To elucidate the molecular mechanism of ClpB-mediated protein disaggregation.
• To investigate the translocation activity and processivity of ClpB.
• To understand the role of the IGL loop in ClpB function and its interaction with ClpP.

Main Methods:

• Single-turnover fluorescence and anisotropy experiments to assess ClpB translocation and dissociation.
• Single-turnover Förster Resonance Energy Transfer (FRET) experiments to study substrate translocation through the axial channel.
• Protein engineering to create ClpB variants with altered functional domains (e.g., IGL loop from ClpA).

Main Results:

• ClpB functions as a non-processive polypeptide translocase, performing one or two translocation steps before dissociation.
• Engineered ClpB with the ClpA IGL loop does not translocate substrate into ClpP for degradation.
• This engineered ClpB variant dysregulates ClpP, causing non-specific proteolysis similar to ADEP dysregulation.

Conclusions:

• ClpB disaggregates proteins through a mechanism involving repeated "tugging and releasing" of exposed polypeptide segments.
• ClpB's translocation is non-processive, with rapid dissociation limiting its ability to thread substrates fully.
• The IGL loop plays a critical role in regulating ClpB's interaction with ClpP and its disaggregation function.