title: Examination of polypeptide substrate specificity for Escherichia coli ClpB.
abstract: Escherichia coli ClpB is a molecular chaperone that belongs to the Clp/Hsp100 family of AAA+ proteins. ClpB is able to form a hexameric ring structure to catalyze protein disaggregation with the assistance of the DnaK chaperone system. Our knowledge of the mechanism of how ClpB recognizes its substrates is still limited. In this study, we have quantitatively investigated ClpB binding to a number of unstructured polypeptides using steady-state anisotropy titrations. To precisely determine the binding affinity for the interaction between ClpB hexamers and polypeptide substrates the titration data were subjected to global non-linear least squares analysis incorporating the dynamic equilibrium of ClpB assembly. Our results show that ClpB hexamers bind tightly to unstructured polypeptides with binding affinities in the range of ∼3-16 nM. ClpB exhibits a modest preference of binding to Peptide B1 with a binding affinity of (1.7 ± 0.2) nM. Interestingly, we found that ClpB binds to an unstructured polypeptide substrate of 40 and 50 amino acids containing the SsrA sequence at the C-terminus with an affinity of (12 ± 3) nM and (4 ± 2) nM, respectively. Whereas, ClpB binds the 11-amino acid SsrA sequence with an affinity of (140 ± 20) nM, which is significantly weaker than other polypeptide substrates that we tested here. We hypothesize that ClpB, like ClpA, requires substrates with a minimum length for optimal binding. Finally, we present evidence showing that multiple ClpB hexamers are involved in binding to polypeptides ≥152 amino acids.
authors.affiliation: Department of Chemistry, The University of Alabama at Birmingham, Birmingham, Alabama, 35294-1240.
authors.forename: Tao
authors.initials: T
authors.lastname: Li
authors.forename: Jiabei
authors.initials: J
authors.lastname: Lin
authors.forename: Aaron L
authors.initials: AL
authors.lastname: Lucius
mesh_terms: D000595:Amino Acid Sequence; D016880:Anisotropy; D002364:Caseins / Q000378:metabolism; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002364:Caseins; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D010455:Peptides; D049071:Endopeptidase Clp; C069775:ClpB protein, E coli
keywords: AAA+ motor proteins; anisotropy; binding; chaperones; fluorescence spectroscopy; protein disaggregation; protein quality control; protein unfoldases; thermodynamics; translocases