Protease Power Strokes Force Proteins to Unfold

Protease Power Strokes Force Proteins to Unfold

Jorge Alegre-Cebollada, Pallav Kosuri, and Julio M. Fernandez

ATP-dependent proteases degrade proteins in the cytosol of cells. Two recent articles, by Aubin-Tam et al. (2011) and Maillard et al. (2011), use single-molecule optical tweezers to show directly that these molecular machines use the energy derived from ATP hydrolysis to mechanically unfold and translocate its substrates into the proteolytic chamber.

DOI

Journal: Cell

Single-Molecule Protein Unfolding and Translocation by an ATP-Fueled Proteolytic Machine

Marie-Eve Aubin-Tam, Adrian O. Olivares, Robert T. Sauer, Tania A. Baker, and Matthew J. Lang

All cells employ ATP-powered proteases for protein-quality control and regulation. In the ClpXP protease, ClpX is a AAA+ machine that recognizes specific protein substrates, unfolds these molecules, and then translocates the denatured polypeptide through a central pore and into ClpP for degradation. Here, we use optical-trapping nanometry to probe the mechanics of enzymatic unfolding and translocation of single molecules of a multidomain substrate. Our experiments demonstrate the capacity of ClpXP and ClpX to perform mechanical work under load, reveal very fast and highly cooperative unfolding of individual substrate domains, suggest a translocation step size of 5–8 amino acids, and support a power-stroke model of denaturation in which successful enzyme-mediated unfolding of stable domains requires coincidence between mechanical pulling by the enzyme and a transient stochastic reduction in protein stability. We anticipate that single-molecule studies of the mechanical properties of other AAA+ proteolytic machines will reveal many shared features with ClpXP.

DOI

Journal: Cell

ClpX(P) Generates Mechanical Force to Unfold and Translocate Its Protein Substrates

Rodrigo A. Maillard, Gheorghe Chistol, Maya Sen, Maurizio Righini, Jiongyi Tan, Christian M. Kaiser, Courtney Hodges, Andreas Martin, and Carlos Bustamante

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.

DOI

Journal: Cell