The ClpXP protease unfolds substrates using a constant rate of pulling but different gears.
ATP-dependent proteases are vital to maintain cellular protein homeostasis. Here, we study the mechanisms of force generation and intersubunit coordination in the ClpXP protease from E. coli to understand how these machines couple ATP hydrolysis to mechanical protein unfolding. Single-molecule analyses reveal that phosphate release is the force-generating step in the ATP-hydrolysis cycle and that ClpXP translocates substrate polypeptides in bursts resulting from highly coordinated conformational changes in two to four ATPase subunits. ClpXP must use its maximum successive firing capacity of four subunits to unfold stable substrates like GFP. The average dwell duration between individual bursts of translocation is constant, regardless of the number of translocating subunits, implying that ClpXP operates with constant "rpm" but uses different "gears."
Jason L. Choy Laboratory of Single-Molecule Biophysics, University of California, Berkeley, CA 94720, USA.|Department of Chemistry, University of California, Berkeley, CA 94720, USA.
Maya
M
Sen
Jason L. Choy Laboratory of Single-Molecule Biophysics, University of California, Berkeley, CA 94720, USA.|QB3 Institute, University of California, Berkeley, CA 94720, USA.
Rodrigo A
RA
Maillard
QB3 Institute, University of California, Berkeley, CA 94720, USA.|Biophysics Graduate Group, University of California, Berkeley, CA 94720, USA.
Kristofor
K
Nyquist
Jason L. Choy Laboratory of Single-Molecule Biophysics, University of California, Berkeley, CA 94720, USA.|QB3 Institute, University of California, Berkeley, CA 94720, USA.|Biophysics Graduate Group, University of California, Berkeley, CA 94720, USA.
Piere
P
Rodriguez-Aliaga
Jason L. Choy Laboratory of Single-Molecule Biophysics, University of California, Berkeley, CA 94720, USA.
Steve
S
Pressé
QB3 Institute, University of California, Berkeley, CA 94720, USA.|Department of Molecular & Cell Biology, University of California, Berkeley, CA 94720, USA.
Andreas
A
Martin
Jason L. Choy Laboratory of Single-Molecule Biophysics, University of California, Berkeley, CA 94720, USA.|QB3 Institute, University of California, Berkeley, CA 94720, USA.|Department of Chemistry, University of California, Berkeley, CA 94720, USA.|Department of Physics, University of California, Berkeley, CA 94720, USA.|Department of Molecular & Cell Biology, University of California, Berkeley, CA 94720, USA.|Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA.
Carlos
C
Bustamante
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D049071:Endopeptidase Clp / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D052898:Optical Tweezers; D010710:Phosphates / Q000378:metabolism; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D058767:Protein Unfolding
D029968:Escherichia coli Proteins; D010710:Phosphates; D021122:Protein Subunits; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; C483573:ClpXP protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases