Conserved residues in the N-domain of the AAA+ chaperone ClpA regulate substrate recognition and unfolding.
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.
Erbse
Annette H
AH
 Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg, Heidelberg, Germany.
Wagner
Judith N
JN
 Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg, Heidelberg, Germany.
Truscott
Kaye N
KN
 Department of Biochemistry, La Trobe University, Melbourne, Australia.
Spall
Sukhdeep K
SK
 Department of Biochemistry, La Trobe University, Melbourne, Australia.
Kirstein
Janine
J
 Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg, Heidelberg, Germany.| Institut für Biologie, Freie Universität Berlin, Berlin, Germany.
Zeth
Kornelius
K
 MPI für Entwicklungsbiologie, Tubingen, Germany.
Turgay
Kürsad
K
 Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg, Heidelberg, Germany.| Institut für Biologie, Freie Universität Berlin, Berlin, Germany.
Mogk
Axel
A
 Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg, Heidelberg, Germany.
Bukau
Bernd
B
 Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg, Heidelberg, Germany.
Dougan
David A
DA
 Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg, Heidelberg, Germany.| Department of Biochemistry, La Trobe University, Melbourne, Australia.
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
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