title: Walker-A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB.
abstract: 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 gamma-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.
authors.affiliation: Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Nagy
authors.forename: Hui-Chuan
authors.initials: HC
authors.lastname: Wu
authors.forename: Zhonghua
authors.initials: Z
authors.lastname: Liu
authors.forename: Sabina
authors.initials: S
authors.lastname: Kedzierska-Mieszkowska
authors.forename: Michal
authors.initials: M
authors.lastname: Zolkiewski
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels; D020816:Amino Acid Motifs / Q000502:physiology*; D019943:Amino Acid Substitution; D001216:Asparagine / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics*; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D020836:Protein Structure, Quaternary; D013912:Threonine / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D013912:Threonine; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D001216:Asparagine; D000255:Adenosine Triphosphate; D005954:Glucosephosphate Dehydrogenase; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli; C069775:ClpB protein, E coli