title: ClpX(P) generates mechanical force to unfold and translocate its protein substrates.
abstract: AAA(+) unfoldases denature and translocate polypeptides into associated peptidases. We report direct observations of mechanical, force-induced protein unfolding by the ClpX unfoldase from E. coli, alone, and in complex with the ClpP peptidase. ClpX hydrolyzes ATP to generate mechanical force and translocate polypeptides through its central pore. Threading is interrupted by pauses that are found to be off the main translocation pathway. ClpX's translocation velocity is force dependent, reaching a maximum of 80 aa/s near-zero force and vanishing at around 20 pN. ClpX takes 1, 2, or 3 nm steps, suggesting a fundamental step-size of 1 nm and a certain degree of intersubunit coordination. When ClpX encounters a folded protein, it either overcomes this mechanical barrier or slips on the polypeptide before making another unfolding attempt. Binding of ClpP decreases the slip probability and enhances the unfolding efficiency of ClpX. Under the action of ClpXP, GFP unravels cooperatively via a transient intermediate.
authors.affiliation: Jason L. Choy Laboratory of Single-Molecule Biophysics, University of California, Berkeley, 94720, USA.
authors.forename: Rodrigo A
authors.initials: RA
authors.lastname: Maillard
authors.forename: Gheorghe
authors.initials: G
authors.lastname: Chistol
authors.forename: Maya
authors.initials: M
authors.lastname: Sen
authors.forename: Maurizio
authors.initials: M
authors.lastname: Righini
authors.forename: Jiongyi
authors.initials: J
authors.lastname: Tan
authors.forename: Christian M
authors.initials: CM
authors.lastname: Kaiser
authors.forename: Courtney
authors.initials: C
authors.lastname: Hodges
authors.forename: Andreas
authors.initials: A
authors.lastname: Martin
authors.forename: Carlos
authors.initials: C
authors.lastname: Bustamante
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001696:Biomechanical Phenomena; D049071:Endopeptidase Clp / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D021381:Protein Transport
chemical_list: D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D049452:Green Fluorescent Proteins; C483572:ClpP protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities