ArticleWalker-A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB  |
Maria Nagy 1 , Hui-Chuan Wu 1, Zhonghua Liu 1 a, Sabina Kedzierska-Mieszkowska 2, Michal Zolkiewski 1 * |
1Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506
2Department of Biochemistry, University of Gdansk, 80-822 Gdansk, Poland
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| email: Michal Zolkiewski (michalz@ksu.edu) |
*Correspondence to Michal Zolkiewski, Department of Biochemistry, 141 Chalmers Hall, Kansas State University, Manhattan, KS 66506
aCurrent address: Department of Embryology, Carnegie Institution, Baltimore, MD 21218
Conflict of interest: The authors declare no competing interests.
Maria Nagy and Hui-Chuan Wu contributed equally to this work.
Funded by:
Dystonia Medical Research Foundation, The National Institutes of Health; Grant Number: P20 RR016475
The Polish Committee for Scientific Research; Grant Number: 0668/P01/2006/30
The Kansas Agricultural Experiment Station; Grant Number: 08-72-J
| AAA+ ATPase molecular chaperone protein aggregation aggregate reactivation site-directed mutagenesis nucleotide binding |
Hexameric AAA+ ATPases induce conformational changes in a variety of macromolecules. AAA+ structures contain the nucleotide-binding P-loop with the Walker A sequence motif: GxxGxGK(T/S). A subfamily of AAA+ sequences contains Asn in the Walker A motif instead of Thr or Ser. This noncanonical subfamily includes torsinA, an ER protein linked to human dystonia and DnaC, a bacterial helicase loader. Role of the noncanonical Walker A motif in the functionality of AAA+ ATPases has not been explored yet. To determine functional effects of introduction of Asn into the Walker A sequence, we replaced the Walker-A Thr with Asn in ClpB, a bacterial AAA+ chaperone which reactivates aggregated proteins. We found that the T-to-N mutation in Walker A partially inhibited the ATPase activity of ClpB, but did not affect the ClpB capability to associate into hexamers. Interestingly, the noncanonical Walker A sequence in ClpB induced preferential binding of ADP vs. ATP and uncoupled the linkage between the ATP-bound conformation and the high-affinity binding to protein aggregates. As a consequence, ClpB with the noncanonical Walker A sequence showed a low chaperone activity in vitro and in vivo. Our results demonstrate a novel role of the Walker-A Thr in sensing the nucleotide's  -phosphate and in maintaining an allosteric linkage between the P-loop and the aggregate binding site of ClpB. We postulate that AAA+ ATPases with the noncanonical Walker A might utilize distinct mechanisms to couple the ATPase cycle with their substrate-remodeling activity. |
Received: 29 August 2008; Accepted: 10 November 2008
10.1002/pro.36
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