title: The molecular chaperone, ClpA, has a single high affinity peptide binding site per hexamer.
abstract: Substrate recognition by Clp chaperones is dependent on interactions with motifs composed of specific peptide sequences. We studied the binding of short motif-bearing peptides to ClpA, the chaperone component of the ATP-dependent ClpAP protease of Escherichia coli in the presence of ATPgammaS and Mg2+ at pH 7.5. Binding was measured by isothermal titration calorimetry (ITC) using the peptide, AANDENYALAA, which corresponds to the SsrA degradation motif found at the C terminus of abnormal nascent polypeptides in vivo. One SsrA peptide was bound per hexamer of ClpA with an association constant (K(A)) of 5 x 10(6) m(-1). Binding was also assayed by changes in fluorescence of an N-terminal dansylated SsrA peptide, which bound with the same stoichiometry of one per ClpA hexamer (K(A) approximately 1 x 10(7) m(-1)). Similar results were obtained when ATP was substituted for ATPgammaS at 6 degrees C. Two additional peptides, derived from the phage P1 RepA protein and the E. coli HemA protein, which bear different substrate motifs, were competitive inhibitors of SsrA binding and bound to ClpA hexamers with K(A)' > 3 x 10(7) m(-1). DNS-SsrA bound with only slightly reduced affinity to deletion mutants of ClpA missing either the N-terminal domain or the C-terminal nucleotide-binding domain, indicating that the binding site for SsrA lies within the N-terminal nucleotide-binding domain. Because only one protein at a time can be unfolded and translocated by ClpA hexamers, restricting the number of peptides initially bound should avoid nonproductive binding of substrates and aggregation of partially processed proteins.
authors.affiliation: Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, MD 20892-8012, USA. grzegorz_piszczek@nih.gov
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Piszczek
authors.forename: Jan
authors.initials: J
authors.lastname: Rozycki
authors.forename: Satyendra K
authors.initials: SK
authors.lastname: Singh
authors.forename: Ann
authors.initials: A
authors.lastname: Ginsburg
authors.forename: Michael R
authors.initials: MR
authors.lastname: Maurizi
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D020816:Amino Acid Motifs; D016880:Anisotropy; D001665:Binding Sites; D001667:Binding, Competitive; D002151:Calorimetry; D004305:Dose-Response Relationship, Drug; D049071:Endopeptidase Clp / Q000737:chemistry* / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000502:physiology*; D017353:Gene Deletion; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D018832:Molecular Chaperones / Q000378:metabolism; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012329:RNA, Bacterial / Q000737:chemistry; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors
chemical_list: D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D010455:Peptides; D012329:RNA, Bacterial; C096222:tmRNA; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; C483568:ClpA protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities