E. coli ClpB is a Robust and Processive Protein Unfoldase
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Motor proteins of the AAA+ (ATPases Associated with Various Cellular Activity) superfamily are responsible for maintenance of the protein crowded environment of the cell. E. coli ClpB is a AAA+ molecular motor that, in collaboration with co-chaperones, couples the energy from ATP hydrolysis to resolving protein aggregates that form during heat shock or stress. ClpB assembles into hexameric rings and, based on structural comparison to known protein translocases or unfoldases, the motor has been proposed to processively unfold proteins and translocate the polypeptide through its axial channel. However, since the translocated polypeptide both enters and leaves the reaction without covalent modification it has proven difficult to test and quantify this model of function. Here we have developed a sequential mixing stopped-flow method that reports on processive protein unfolding and translocation catalyzed by ClpB. Using this approach, we show that ClpB processively and sequentially unfolds up to three tandem repeats of the stably folded Titin I27 domain with a maximum length of 362 amino acids. We report that ClpB unfolds ∼60 amino acids between two rate limiting steps at a rate of ∼0.9 aa s -1 in the presence of a 1:1 mixture of ATP:ATPγS. In contrast to what was previously reported by others, in the absence of ATP, we show that ClpB efficiently couples ATPγS hydrolysis to processive unfolding at a rate of ∼0.09 aa s -1 . Our approach solves the problem of needing covalent modification of the substrate to detect translocation. Thus, we have opened the door to addressing many questions regarding AAA+ protein function that is desperately needed to complement and test the wealth of new structural information emerging from cryo-EM studies on these motors.
Significance Statement
AAA+ molecular chaperones are essential for proteome maintenance across domains of life. Bacterial ClpB has the unique ability to disrupt protein aggregates that form during heat shock or stress. Although ClpB has been hypothesized to disrupt aggregates by processively unfolding and translocating proteins, this model has proven difficult to test and quantify. This is, in part, because the protein substrate enters and leaves the reaction without covalent modification. We have overcome the difficulty of detecting translocation without modification by developing a sequential mixing stopped-flow strategy sensitive to arrival of a motor at a fixed position on the enzymatically unfolded polypeptide. We show that E. coli ClpB is a robust and processive protein unfoldase. This work will allow us to address many questions regarding AAA+ protein function that were previously inaccessible.
