Hexameric AAA1 ATPases induce conformational changes in a variety of macromolecules. AAA1 structures contain the nucleotide-binding P-loop with the Walker A sequence motif: GxxGxGK(T/S). A subfamily of AAA1 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 AAA1 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 AAA1 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 c-phosphate and in maintaining an allosteric linkage between the P-loop and the aggregate binding site of ClpB. We postulate that AAA1 ATPases with the noncanonical Walker A might utilize distinct mechanisms to couple the ATPase cycle with their substrate-remodeling activity.
Dystonia is a disease characterized by involuntary and sustained muscle contractions that lead to paralysis and abnormal postures. The most severe type of dystonia, early‐onset Torsion dystonia, is associated with a deletion of a glutamate (ΔE) near the C‐terminus in TorsinA. TorsinA is an endoplasmic reticulum (ER) AAA+ ATPase of ill‐defined function, and the reasons that the ΔE variant is associated with the disease are unclear. Further, the penetrance of the disease is highly variable, suggesting that genetic modifiers affect disease severity. In order to identify modifiers of TorsinA stability and folding, we developed a TorsinA expression system in Saccharomyces cerevisiae. Similar to TorsinA in mammalian cells, TorsinA and TorsinAΔE in yeast associate with the luminal side of the ER membrane and are glycosylated. We found that the stability and glycosylation of TorsinA and TorsinAΔE depend on the yeast ER Hsp70 chaperone BiP and Hsp40 co‐chaperones Jem1 and Scj1. Mutations in TorsinA that prevent glycosylation, ATP binding, or ATP hydrolysis, or that eliminate the hydrophobic domain led to BiP‐dependent degradation, and to differences in protease sensitivity and membrane association. Therefore, we have identified conserved ER chaperones that are critical for TorsinA conformation and stability. We are testing these findings in mammalian systems. Supported by the Dystonia Medical Research Foundation.
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