title: Stochastic but highly coordinated protein unfolding and translocation by the ClpXP proteolytic machine.
abstract: ClpXP and other AAA+ proteases recognize, mechanically unfold, and translocate target proteins into a chamber for proteolysis. It is not known whether these remarkable molecular machines operate by a stochastic or sequential mechanism or how power strokes relate to the ATP-hydrolysis cycle. Single-molecule optical trapping allows ClpXP unfolding to be directly visualized and reveals translocation steps of ∼1-4 nm in length, but how these activities relate to solution degradation and the physical properties of substrate proteins remains unclear. By studying single-molecule degradation using different multidomain substrates and ClpXP variants, we answer many of these questions and provide evidence for stochastic unfolding and translocation. We also present a mechanochemical model that accounts for single-molecule, biochemical, and structural results for our observation of enzymatic memory in translocation stepping, for the kinetics of translocation steps of different sizes, and for probabilistic but highly coordinated subunit activity within the ClpX ring.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN 37235, USA.
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Cordova
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Adrian O
authors.initials: AO
authors.lastname: Olivares
authors.affiliation: Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Yongdae
authors.initials: Y
authors.lastname: Shin
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Benjamin M
authors.initials: BM
authors.lastname: Stinson
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Stephane
authors.initials: S
authors.lastname: Calmat
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Karl R
authors.initials: KR
authors.lastname: Schmitz
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, the Netherlands.
authors.forename: Marie-Eve
authors.initials: ME
authors.lastname: Aubin-Tam
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Tania A
authors.initials: TA
authors.lastname: Baker
authors.affiliation: Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN 37235, USA; Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37235, USA. Electronic address: matt.lang@vanderbilt.edu.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Lang
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: bobsauer@mit.edu.
authors.forename: Robert T
authors.initials: RT
authors.lastname: Sauer
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D049071:Endopeptidase Clp / Q000737:chemistry* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D059748:Proteolysis
chemical_list: D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; C483572:ClpP protease, E coli; C568646:ClpP protein, human; D049071:Endopeptidase Clp; C528384:CLPX protein, human
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