Dissection of Axial-Pore Loop Function during Unfolding and Translocation by a AAA+ Proteolytic Machine.

Area of Science:

• Molecular biology
• Biochemistry
• Protein dynamics

Background:

• ClpX and related AAA+ proteins are hexameric machines that remodel protein substrates.
• The pore-1 loops within these machines are hypothesized to be crucial for substrate engagement, unfolding, and translocation.
• The precise roles of these loops in substrate interaction and processivity remain unclear.

Purpose of the Study:

• To investigate the specific functions of ClpX pore-1 loops in protein substrate processing.
• To determine whether pore-1 loops are involved in preventing substrate dissociation during translocation.
• To elucidate the mechanism by which ClpX grips and unfolds protein substrates.

Main Methods:

• Utilized ClpX pore-1-loop variants to probe loop function.
• Employed single-molecule force spectroscopy to measure mechanical interactions.
• Conducted ensemble assays to assess overall enzymatic activity and substrate processing.

Main Results:

• The six pore-1 loops function synchronously to grip and unfold substrates during a mechanical power stroke.
• Pore-1 loops are not critical for preventing substrate slipping between power strokes.
• ClpX variants with mutated pore-1 loops exhibited unfolding defects and increased substrate release, particularly for mechanically stable proteins.

Conclusions:

• ClpX pore-1 loops are essential for the mechanical unfolding of protein substrates.
• Substrate gripping strength, mediated by pore-1 loops, is crucial for efficient unfolding but not for preventing slippage.
• A threshold model for substrate gripping explains the observed defects in mutant ClpX variants.