Stepwise Unfolding of a β Barrel Protein by the AAA+ ClpXP Protease
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Sauer_Stepwise unfolding.pdf
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Author(s)
Nager, Andrew Ross
Sauer, Robert T.
Baker, Tania
Date Issued
July 2011
Journal
Journal of Molecular Biology
Publisher
Elsevier
Citation
Nager, Andrew R., Tania A. Baker, and Robert T. Sauer. “Stepwise Unfolding of a β Barrel Protein by the AAA+ ClpXP Protease.” Journal of Molecular Biology 413, no. 1 (October 2011): 4–16.
Version
Author's final manuscript
Abstract
In the AAA+ ClpXP protease, ClpX uses the energy of ATP binding and hydrolysis to unfold proteins before translocating them into ClpP for degradation. For proteins with C-terminal ssrA tags, ClpXP pulls on the tag to initiate unfolding and subsequent degradation. Here, we demonstrate that an initial step in ClpXP unfolding of the 11-stranded β barrel of superfolder GFP-ssrA involves extraction of the C-terminal β strand. The resulting 10-stranded intermediate is populated at low ATP concentrations, which stall ClpXP unfolding, and at high ATP concentrations, which support robust degradation. To determine if stable unfolding intermediates cause low-ATP stalling, we designed and characterized circularly permuted GFP variants. Notably, stalling was observed for a variant that formed a stable 10-stranded intermediate but not for one in which this intermediate was unstable. A stepwise degradation model in which the rates of terminal-strand extraction, strand refolding or recapture, and unfolding of the 10-stranded intermediate all depend on the rate of ATP hydrolysis by ClpXP accounts for the observed changes in degradation kinetics over a broad range of ATP concentrations. Our results suggest that the presence or absence of unfolding intermediates will play important roles in determining whether forced enzymatic unfolding requires a minimum rate of ATP hydrolysis.
MIT Department
Massachusetts Institute of Technology. Department of Biology
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DOI of Published Version
https://doi.org/10.1016/j.jmb.2011.07.041
