title: The amino-terminal domain of ClpB supports binding to strongly aggregated proteins.
abstract: Bacterial heat-shock proteins, ClpB and DnaK form a bichaperone system that efficiently reactivates aggregated proteins. ClpB undergoes nucleotide-dependent self-association and forms ring-shaped oligomers. The ClpB-assisted dissociation of protein aggregates is linked to translocation of substrates through the central channel in the oligomeric ClpB. Events preceding the translocation step, such as recognition of aggregates by ClpB, have not yet been explored, and the location of the aggregate-binding site in ClpB has been under discussion. We investigated the reactivation of aggregated glucose-6-phosphate dehydrogenase (G6PDH) by ClpB and its N-terminally truncated variant ClpBDeltaN in the presence of DnaK, DnaJ, and GrpE. We found that the chaperone activity of ClpBDeltaN becomes significantly lower than that of the full-length ClpB as the size of G6PDH aggregates increases. Using a "substrate trap" variant of ClpB with mutations of Walker B motifs in both ATP-binding modules (E279Q/E678Q), we demonstrated that ClpBDeltaN binds to G6PDH aggregates with a significantly lower affinity than the full-length ClpB. Moreover, we identified two conserved acidic residues at the surface of the N-terminal domain of ClpB that support binding to G6PDH aggregates. Those N-terminal residues (Asp-103, Glu-109) contribute as much substrate-binding capability to ClpB as the conserved Tyr located at the entrance to the ClpB channel. In summary, we provided evidence for an essential role of the N-terminal domain of ClpB in recognition and binding strongly aggregated proteins.
authors.affiliation: Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
authors.forename: Micheal E
authors.initials: ME
authors.lastname: Barnett
authors.forename: Maria
authors.initials: M
authors.lastname: Nagy
authors.forename: Sabina
authors.initials: S
authors.lastname: Kedzierska
authors.forename: Michal
authors.initials: M
authors.lastname: Zolkiewski
mesh_terms: D020816:Amino Acid Motifs; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D049071:Endopeptidase Clp; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013997:Time Factors
chemical_list: D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D005954:Glucosephosphate Dehydrogenase; D049071:Endopeptidase Clp; C069775:ClpB protein, E coli