E. coli ClpB Exhibits Processive Unfolding and Translocation of Stably Folded Protein Substrates

Temel Kavramlar

E. coli ClpB, a protein disaggregase, can processively unfold and translocate stably folded protein substrates through its hexameric ring structure, with a kinetic step-size of ~60 amino acids and an overall rate of ~0.9 amino acids per second.

Özet

The content describes the development of a single-turnover stopped-flow fluorescence method to study the mechanism of protein unfolding and translocation catalyzed by the E. coli protein disaggregase ClpB. Key findings:

1. ClpB can processively unfold and translocate stably folded protein substrates, such as tandem repeats of the Titin I27 domain, through its hexameric ring structure.
2. The kinetic step-size, or the average number of amino acids unfolded between two rate-limiting steps, is ~60 amino acids.
3. The overall rate of unfolding and translocation is ~0.9 amino acids per second in the presence of a 1:1 mixture of ATP and the slowly hydrolyzable ATP analog ATPγS.
4. Reducing the ATPγS concentration increases the overall rate to ~4.3 amino acids per second, suggesting that protein unfolding is rate-limiting and translocation is much faster.
5. The authors propose a model where slow unfolding is followed by fast translocation, allowing ClpB to rapidly translocate up to the next folded region or rapidly dissociate if no additional fold is encountered.
6. This stopped-flow method overcomes the limitation of needing covalent modification to detect enzyme-catalyzed protein unfolding and translocation, and can be broadly applied to other AAA+ molecular motors.

İstatistikler

The content includes the following key data:

ClpB translocates at a rate of ~0.9 amino acids per second in the presence of a 1:1 mixture of ATP and ATPγS.
ClpB translocates at a rate of ~4.3 amino acids per second in the presence of a ~3:1 mixture of ATP and ATPγS.
The kinetic step-size, or the average number of amino acids unfolded between two rate-limiting steps, is ~57-58 amino acids.

Alıntılar

"We propose a model where slow unfolding followed by fast translocation represents an important mechanistic feature that allows the motor to rapidly translocate up to the next folded region or rapidly dissociate if no additional fold is encountered."

Önemli Bilgiler Şuradan Elde Edildi

E. coli ClpB is a Robust and Processive Protein Unfoldase

by Banwait,J. K... : www.biorxiv.org 03-19-2024

https://www.biorxiv.org/content/10.1101/2024.03.18.584833v2

Daha Derin Sorular

What are the potential implications of the slow unfolding and fast translocation mechanism of ClpB on its role in protein disaggregation and proteome maintenance

The slow unfolding and fast translocation mechanism of ClpB can have significant implications for its role in protein disaggregation and proteome maintenance. Firstly, the ability of ClpB to unfold proteins at a slow rate of approximately 0.9 amino acids per second before rapidly translocating the unfolded polypeptide chain suggests a highly efficient and controlled process. This mechanism allows ClpB to carefully unfold proteins in a stepwise manner, ensuring proper processing and preventing protein misfolding or aggregation. The fast translocation rate of up to 4.3 amino acids per second enables ClpB to quickly move the unfolded protein through its axial channel, facilitating the disaggregation process.
In terms of protein disaggregation, the slow unfolding step ensures thorough and accurate processing of misfolded or aggregated proteins, while the rapid translocation step allows for efficient removal of these proteins from aggregates. This mechanism is crucial for maintaining protein homeostasis and preventing the accumulation of toxic protein aggregates that can lead to cellular dysfunction and disease. Additionally, the processive nature of ClpB's unfolding and translocation mechanism allows it to handle a wide range of substrates efficiently, contributing to its role in proteome maintenance.
Overall, the slow unfolding and fast translocation mechanism of ClpB ensures precise and efficient protein disaggregation, contributing to the maintenance of a functional proteome and cellular health.

How might the kinetic step-size and overall rate of unfolding and translocation be influenced by the stability and structural features of the folded protein substrates

The kinetic step-size and overall rate of unfolding and translocation by ClpB can be influenced by the stability and structural features of the folded protein substrates. The kinetic step-size, which represents the average number of amino acids unfolded or translocated between two rate-limiting steps, may vary depending on the stability of the folded protein structure.
For more stable and structured proteins, the kinetic step-size may be larger as ClpB needs to overcome stronger interactions and structural barriers to unfold the protein. This could result in a slower unfolding rate and a larger step-size as ClpB navigates through the folded regions. On the other hand, less stable or unstructured proteins may have a smaller kinetic step-size as ClpB can more easily unfold and translocate through these regions, leading to a faster overall rate of unfolding and translocation.
Additionally, the overall rate of unfolding and translocation may be influenced by the structural features of the folded protein substrates. Proteins with complex or compact structures may require more time and energy for ClpB to unfold and translocate, resulting in a slower overall rate. In contrast, proteins with simpler or more accessible structures may be processed more quickly by ClpB, leading to a faster overall rate of unfolding and translocation.
Therefore, the stability and structural complexity of the folded protein substrates can impact the kinetic step-size and overall rate of unfolding and translocation by ClpB, highlighting the importance of substrate characteristics in the protein disaggregation process.

Could the stopped-flow method developed in this study be adapted to investigate the unfolding and translocation mechanisms of other AAA+ molecular motors that do not covalently modify their substrates

The stopped-flow method developed in this study could be adapted to investigate the unfolding and translocation mechanisms of other AAA+ molecular motors that do not covalently modify their substrates. By modifying the substrates to include fluorescent labels or other detection methods, researchers can apply the same single turnover stopped-flow strategy to study the unfolding and translocation kinetics of different AAA+ motors.
The method's ability to detect processive protein unfolding and translocation without the need for covalent modification makes it a valuable tool for studying the mechanisms of various molecular motors. Researchers can use this approach to explore the kinetics, step-sizes, and processivity of other AAA+ motors, providing insights into their roles in cellular processes such as proteome maintenance, DNA replication, and protein quality control.
Furthermore, by adapting the stopped-flow method to different AAA+ motors, researchers can compare and contrast the unfolding and translocation mechanisms across various motor proteins, leading to a better understanding of their functional diversity and regulatory mechanisms. This approach can contribute to uncovering the fundamental principles governing AAA+ motor activity and their impact on cellular physiology.