title: ATPγS competes with ATP for binding at Domain 1 but not Domain 2 during ClpA catalyzed polypeptide translocation.
abstract: ClpAP is an ATP-dependent protease that assembles through the association of hexameric rings of ClpA with the cylindrically-shaped protease ClpP. ClpA contains two nucleotide binding domains, termed Domain 1 (D1) or 2 (D2). We have proposed that D1 or D2 limits the rate of ClpA catalyzed polypeptide translocation when ClpP is either absent or present, respectively. Here we show that the rate of ClpA catalyzed polypeptide translocation depends on [ATPγS] in the absence of ClpP, but not in the presence of ClpP. We observe that ATPγS non-cooperatively binds to ClpA during polypeptide translocation with an apparent affinity of ~6 μM, but that introduction of ClpP shifts this affinity such that translocation is not affected. Interpreting these data with our proposed model for translocation catalyzed by ClpA vs. ClpAP suggests that ATPγS competes for binding at D1 but not at D2.
authors.affiliation: Department of Chemistry, The University of Alabama at Birmingham, 1530 3rd Ave S, Birmingham, AL 35294-1240, United States.
authors.forename: Justin M
authors.initials: JM
authors.lastname: Miller
authors.affiliation: Department of Chemistry, The University of Alabama at Birmingham, 1530 3rd Ave S, Birmingham, AL 35294-1240, United States. Electronic address: allucius@uab.edu.
authors.forename: Aaron L
authors.initials: AL
authors.lastname: Lucius
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D049071:Endopeptidase Clp / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary
chemical_list: D029968:Escherichia coli Proteins; D010455:Peptides; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; C483568:ClpA protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases
keywords: AAA+ motor proteins; ATP dependent proteases; Pre-steady-state kinetics; Protein unfoldases; Steady-state kinetics