ClpB chaperone passively threads soluble denatured proteins through its central pore

Area of Science:

• Molecular Biology
• Protein Degradation
• Biochemistry

Background:

• ClpB disaggregase is a hexameric protein machine that uses ATP to thread substrate proteins through a central pore.
• Each ClpB protomer has two ATP-binding AAA+ modules crucial for its function.
• Understanding the specific roles of these AAA+ modules in protein threading is essential.

Purpose of the Study:

• To investigate the distinct roles of the two AAA+ modules in ClpB-mediated substrate protein threading.
• To determine how mutations in conserved motifs of the AAA+ modules affect the kinetics of protein threading.

Main Methods:

• Utilized an engineered ClpB variant (BAP) capable of binding the ClpP protease.
• Introduced conserved motif mutations into the two AAA+ modules of BAP.
• Measured steady-state threading rates of soluble denatured proteins using Michaelis-Menten kinetics.

Main Results:

• Kinetic parameters (kcat, Km) for substrate threading correlated with mutation type, not solely ATPase activity.
• Some ClpB mutants with minimal or no ATPase activity still exhibited significant protein threading capabilities.
• This suggests that ATP hydrolysis is not the sole driver for threading soluble denatured proteins.

Conclusions:

• ClpB disaggregase possesses a passive threading mechanism for soluble denatured proteins.
• The two AAA+ modules play differential roles in substrate threading, independent of their ATPase activity.
• These findings offer new insights into the mechanical aspects of protein disaggregation by ClpB.