Structural basis for substrate gripping and translocation by the ClpB AAA+ disaggregase

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• Bacterial ClpB and yeast Hsp104 are Hsp100 protein disaggregases crucial for maintaining proteostasis.
• These proteins solubilize protein aggregates by translocating polypeptides through a central channel powered by two AAA+ nucleotide-binding domains (NBDs).
• Understanding their translocation mechanism is key to comprehending protein aggregate disaggregation.

Purpose of the Study:

• To elucidate the molecular mechanism of protein substrate translocation by bacterial ClpB.
• To visualize the distinct interactions involved in substrate gripping and translocation.
• To reveal how ATP hydrolysis drives the directional, stepwise disaggregation cycle.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was used to determine structures.
• A hyperactive ClpB variant was employed.
• Structures were obtained with a model substrate (casein) and a slowly hydrolysable ATP analog (ATPγS).

Main Results:

• Cryo-EM structures revealed distinct substrate-gripping interactions mediated by NBD1 and NBD2 pore loops.
• A trimer of N-terminal domains forms a channel entrance that binds the polypeptide substrate near the NBD1 contact site.
• Conformational changes at the NBD seam interface illustrate ATP hydrolysis-driven substrate engagement and disengagement.

Conclusions:

• The study reveals the detailed mechanism of protein disaggregation by ClpB.
• Distinct NBD pore loops and conformational dynamics are critical for directional substrate translocation.
• This work provides insights into the fundamental process of protein aggregate solubilization by Hsp100 chaperones.