title: A context-dependent ClpX recognition determinant located at the C terminus of phage Mu repressor.
abstract: The bacteriophage Mu immunity repressor is a conformationally sensitive sensor that can be interconverted between forms resistant to and sensitive to degradation by ClpXP protease. Protease-sensitive repressor molecules with an altered C-terminal sequence promote rapid degradation of the wild-type repressor by inducing its C-terminal end to become exposed. Here we determined that the last 5 C-terminal residues (CTD5) of the wild-type repressor contain the motif required for recognition by the ClpX molecular chaperone, a motif that is strongly dependent upon the context in which it is presented. Although attachment of the 11-residue ssrA degradation tag to the C terminus of green fluorescent protein (GFP) promoted its rapid degradation by ClpXP, attachment of 5-27 C-terminal residues of the repressor failed to promote degradation. Disordered peptides derived from 41 and 35 C-terminal residues of CcdA (CcdA41) and thioredoxin (TrxA35), respectively, activated CTD5 when placed as linkers between GFP and repressor C-terminal sequences. However, when the entire thioredoxin sequence was included as a linker to promote an ordered configuration of the TrxA35 peptide, the resulting substrate was not degraded. In addition, a hybrid tag, in which CTD5 replaced the 3-residue recognition motif of the ssrA tag, was inactive when attached directly to GFP but active when attached through the CcdA41 peptide. Thus, CTD5 is sufficient to act as a recognition motif but has requirements for its presentation not shared by the ssrA tag. We suggest that activation of CTD5 may require presentation on a disordered or flexible domain that confers ligand flexibility.
authors.affiliation: Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D.C. 20057, USA.
authors.forename: Dawn A
authors.initials: DA
authors.lastname: Defenbaugh
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nakai
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000502:physiology*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D010583:Bacteriophage mu / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D049071:Endopeptidase Clp; D010450:Endopeptidases / Q000737:chemistry; D029968:Escherichia coli Proteins; D049452:Green Fluorescent Proteins; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013879:Thioredoxins / Q000737:chemistry; D013997:Time Factors; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D001426:Bacterial Proteins; C104882:CcdA protein, Bacillus; D029968:Escherichia coli Proteins; D008024:Ligands; D008164:Luminescent Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D049452:Green Fluorescent Proteins; D013879:Thioredoxins; D010450:Endopeptidases; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities