Stepwise Unfolding of a β Barrel Protein by the AAA plus ClpXP Protease

JOURNAL OF MOLECULAR BIOLOGY

卷:413

Article

Nager, Andrew R.¹  Baker, Tania A.^1,2  Sauer, Robert T.¹ 

[1] MIT, Dept Biol, Cambridge, MA 02139 USA

[2] MIT, Howard Hughes Med Inst, Cambridge, MA 02139 USA

关键词: ATP-dependent degradation;    ClpXP;    enzymatic protein unfolding;    circularly permuted GFP;   

DOI  :  10.1016/j.jmb.2011.07.041

来源: Elsevier

【 摘 要 】

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 beta barrel of superfolder GFP-ssrA involves extraction of the C-terminal beta 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-AT? 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. (C) 2011 Elsevier Ltd. All rights reserved.

【 授权许可】

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