A mutant chaperonin with rearranged inter-ring electrostatic contacts and temperature-sensitive dissociation

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• The chaperonin GroEL is essential for protein folding in Escherichia coli.
• GroEL functions via ATP-dependent, cooperative movements between its two rings, creating folding chambers.
• A specific mutation, E461K, at the inter-ring interface causes temperature-sensitive protein folding defects.

Purpose of the Study:

• To elucidate the molecular mechanism underlying the temperature-sensitive protein folding defect caused by the E461K mutation in GroEL.
• To investigate the structural and functional consequences of the E461K substitution on GroEL's cooperative function.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was employed to examine the structure of the mutant GroEL chaperonin.
• Analysis focused on the alignment of subunit contacts between the two rings and the binding of the cochaperonin GroES.

Main Results:

• The E461K mutation altered the normal out-of-register alignment of inter-ring subunit contacts to an in-register configuration.
• This structural change resulted in a loss of cooperativity in ATP binding and hydrolysis.
• The cochaperonin GroES bound simultaneously to both rings of the E461K mutant, indicating a loss of negative cooperativity.
• GroES-bound E461K complexes were unstable at higher temperatures, dissociating into single-ring complexes that became trapped.

Conclusions:

• The E461K mutation disrupts the allosteric communication between the two rings of GroEL.
• This disruption leads to impaired ATP-dependent cooperativity and the formation of non-productive, dead-end states.
• Understanding these structural and functional defects provides insight into the essential role of GroEL in protein homeostasis.