AAA+ protease-adaptor structures reveal altered conformations and ring specialization

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• ClpAP is a crucial two-ring AAA+ protease in Escherichia coli responsible for degrading proteins targeted by the N-end rule pathway.
• The ClpS adaptor protein is essential for delivering these N-end rule substrates to the ClpAP protease for degradation.

Purpose of the Study:

• To elucidate the high-resolution structural mechanisms of ClpAPS complex assembly and substrate recognition.
• To understand the role of ClpA pore loops and the ClpS N-terminal extension (NTE) in substrate binding and processing.
• To investigate the functional asymmetry between the D1 and D2 rings of ClpA during proteolysis.

Main Methods:

• High-resolution cryo-electron microscopy (cryo-EM) to determine the structures of ClpAPS complexes.
• Biochemical kinetic studies to analyze the protein remodeling activity of ClpA.
• Structural analysis of ClpA pore loop interactions with the ClpS NTE.

Main Results:

• Cryo-EM structures revealed specific interactions between ClpA pore-1 and pore-2 loops and the ClpS NTE, mimicking substrate binding in AAA+ unfoldases.
• Kinetic data indicated that ClpA D1 ring pore-2 loops are critical for catalyzing protein remodeling and substrate delivery by ClpS.
• A distinct structural class showed asymmetric engagement, with D2 pore-1 loops tucked away, suggesting differential roles for the D1 and D2 rings.

Conclusions:

• The ClpAPS complex employs intricate pore loop interactions for substrate recognition and processing, guided by the ClpS adaptor.
• The observed asymmetry in ClpA ring engagement highlights novel functional mechanisms within AAA+ proteases.
• Pore-loop tucking represents a conserved structural motif in AAA+ unfoldases, potentially involved in enzyme regulation like pausing or unloading.