Multistep substrate binding and engagement by the AAA+ ClpXP protease.

Area of Science:

• Biochemistry
• Molecular Biology
• Proteostasis

Background:

• Escherichia coli ClpXP is a well-studied AAA+ protease.
• Mechanisms of substrate binding and engagement by ClpXP are not fully understood.
• Understanding these steps is crucial for elucidating ATP-fueled degradation pathways.

Purpose of the Study:

• To investigate the kinetics and mechanisms of substrate binding to E. coli ClpXP.
• To characterize the distinct substrate-bound complexes formed during ClpXP-substrate interaction.
• To elucidate the role of ATP hydrolysis in substrate engagement and processing.

Main Methods:

• Utilized a fluorescence-quenching assay to monitor substrate binding.
• Employed polyphasic stopped-flow kinetics to determine association and dissociation rates.
• Integrated kinetic data with existing cryo-electron microscopy (cryo-EM) structures.

Main Results:

• Identified at least three distinct substrate-bound complexes (recognition, intermediate, engaged).
• Demonstrated that initial substrate association and dissociation are ATP-independent.
• Showed that ATP hydrolysis accelerates subsequent kinetic steps, facilitating substrate unfolding.
• Proposed a model where the ssrA degron translocates within the ClpX channel, pulling the substrate.

Conclusions:

• ClpXP employs a multi-step binding mechanism involving reversible initial recognition for specificity.
• Subsequent engagement steps ensure robust gripping of diverse substrates for efficient unfolding and degradation.
• This mechanism allows ClpXP to act as a highly specific and versatile protease.