title: Escherichia coli ClpB is a non-processive polypeptide translocase.
abstract: Escherichia coli caseinolytic protease (Clp)B is a hexameric AAA+ [expanded superfamily of AAA (ATPase associated with various cellular activities)] enzyme that has the unique ability to catalyse protein disaggregation. Such enzymes are essential for proteome maintenance. Based on structural comparisons to homologous enzymes involved in ATP-dependent proteolysis and clever protein engineering strategies, it has been reported that ClpB translocates polypeptide through its axial channel. Using single-turnover fluorescence and anisotropy experiments we show that ClpB is a non-processive polypeptide translocase that catalyses disaggregation by taking one or two translocation steps followed by rapid dissociation. Using single-turnover FRET experiments we show that ClpB containing the IGL loop from ClpA does not translocate substrate through its axial channel and into ClpP for proteolytic degradation. Rather, ClpB containing the IGL loop dysregulates ClpP leading to non-specific proteolysis reminiscent of ADEP (acyldepsipeptide) dysregulation. Our results support a molecular mechanism where ClpB catalyses protein disaggregation by tugging and releasing exposed tails or loops.
authors.affiliation: Department of Chemistry, The University of Alabama at Birmingham (UAB), Birmingham AL, U.S.A.
authors.forename: Tao
authors.initials: T
authors.lastname: Li
authors.affiliation: Department of Chemistry, The University of Alabama at Birmingham (UAB), Birmingham AL, U.S.A.
authors.forename: Clarissa L
authors.initials: CL
authors.lastname: Weaver
authors.affiliation: Department of Chemistry, The University of Alabama at Birmingham (UAB), Birmingham AL, U.S.A.
authors.forename: Jiabei
authors.initials: J
authors.lastname: Lin
authors.affiliation: Department of Chemistry, The University of Alabama at Birmingham (UAB), Birmingham AL, U.S.A.
authors.forename: Elizabeth C
authors.initials: EC
authors.lastname: Duran
authors.affiliation: Department of Chemistry, The University of Alabama at Birmingham (UAB), Birmingham AL, U.S.A.
authors.forename: Justin M
authors.initials: JM
authors.lastname: Miller
authors.affiliation: Department of Chemistry, The University of Alabama at Birmingham (UAB), Birmingham AL, U.S.A. allucius@uab.edu.
authors.forename: Aaron L
authors.initials: AL
authors.lastname: Lucius
mesh_terms: D000595:Amino Acid Sequence; D018988:Bacterial Translocation / Q000502:physiology; D049071:Endopeptidase Clp; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary
chemical_list: D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D010455:Peptides; D049071:Endopeptidase Clp; C069775:ClpB protein, E coli
keywords: AAA+ motor proteins; Förster resonance energy transfer (FRET); chaperones; pre-steady-state kinetics; protein disaggregation