title: Conserved residues in the N-domain of the AAA+ chaperone ClpA regulate substrate recognition and unfolding.
abstract: Protein degradation in the cytosol of Escherichia coli is carried out by a variety of different proteolytic machines, including ClpAP. The ClpA component is a hexameric AAA+ (ATPase associated with various cellular activities) chaperone that utilizes the energy of ATP to control substrate recognition and unfolding. The precise role of the N-domains of ClpA in this process, however, remains elusive. Here, we have analysed the role of five highly conserved basic residues in the N-domain of ClpA by monitoring the binding, unfolding and degradation of several different substrates, including short unstructured peptides, tagged and untagged proteins. Interestingly, mutation of three of these basic residues within the N-domain of ClpA (H94, R86 and R100) did not alter substrate degradation. In contrast mutation of two conserved arginine residues (R90 and R131), flanking a putative peptide-binding groove within the N-domain of ClpA, specifically compromised the ability of ClpA to unfold and degrade selected substrates but did not prevent substrate recognition, ClpS-mediated substrate delivery or ClpP binding. In contrast, a highly conserved tyrosine residue lining the central pore of the ClpA hexamer was essential for the degradation of all substrate types analysed, including both folded and unstructured proteins. Taken together, these data suggest that ClpA utilizes two structural elements, one in the N-domain and the other in the pore of the hexamer, both of which are required for efficient unfolding of some protein substrates.
authors.affiliation:  Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg, Heidelberg, Germany.
authors.forename: Annette H
authors.initials: AH
authors.lastname: Erbse
authors.affiliation:  Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg, Heidelberg, Germany.
authors.forename: Judith N
authors.initials: JN
authors.lastname: Wagner
authors.affiliation:  Department of Biochemistry, La Trobe University, Melbourne, Australia.
authors.forename: Kaye N
authors.initials: KN
authors.lastname: Truscott
authors.affiliation:  Department of Biochemistry, La Trobe University, Melbourne, Australia.
authors.forename: Sukhdeep K
authors.initials: SK
authors.lastname: Spall
authors.affiliation:  Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg, Heidelberg, Germany.| Institut für Biologie, Freie Universität Berlin, Berlin, Germany.
authors.forename: Janine
authors.initials: J
authors.lastname: Kirstein
authors.affiliation:  MPI für Entwicklungsbiologie, Tubingen, Germany.
authors.forename: Kornelius
authors.initials: K
authors.lastname: Zeth
authors.affiliation:  Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg, Heidelberg, Germany.| Institut für Biologie, Freie Universität Berlin, Berlin, Germany.
authors.forename: Kürsad
authors.initials: K
authors.lastname: Turgay
authors.affiliation:  Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg, Heidelberg, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Mogk
authors.affiliation:  Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg, Heidelberg, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Bukau
authors.affiliation:  Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg, Heidelberg, Germany.| Department of Biochemistry, La Trobe University, Melbourne, Australia.
authors.forename: David A
authors.initials: DA
authors.lastname: Dougan
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics; D017124:Conserved Sequence; D049071:Endopeptidase Clp / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000235:genetics; D018832:Molecular Chaperones / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D011489:Protein Denaturation / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D001120:Arginine; C483568:ClpA protease, E coli; D049071:Endopeptidase Clp; C069775:ClpB protein, E coli