trans-Targeting of the Phage Mu Repressor Is Promoted by Conformational Changes That Expose Its ClpX Recognition Determinant

Marshall‐Batty, Kimberly R.; Nakai, Hiroshi

doi:10.1074/jbc.m209352200

Cited by 13 publications

(21 citation statements)

References 46 publications

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“…Peptide degradation sequences may be constitutively recognized or become accessible to ClpX only after cleavage by another protease or after a conformational change (11,12). After recognition of the peptide degradation signal by the ClpX processing site, ATP-dependent conformational changes in ClpX are thought to generate a transient ''pulling'' force that destabilizes the attached native protein (13).…”

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confidence: 99%

Distinct peptide signals in the UmuD and UmuD′ subunits of UmuD/D′ mediate tethering and substrate processing by the ClpXP protease

Neher

Sauer

Baker

2003

Proc. Natl. Acad. Sci. U.S.A.

112

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The Escherichia coli UmuD protein is a component of DNA polymerase V, an error-prone polymerase that carries out translesion synthesis on damaged DNA templates. The intracellular concentration of UmuD is strictly controlled by regulated transcription, by posttranslational processing of UmuD to UmuD, and by ClpXP degradation. UmuD is a substrate for the ClpXP protease but must form a heterodimer with its unabbreviated precursor, UmuD, for efficient degradation to occur. Here, we show that UmuD functions as a UmuD delivery protein for ClpXP. UmuD can also deliver a UmuD partner for degradation. UmuD resembles SspB, a wellcharacterized substrate-delivery protein for ClpX, in that both proteins use related peptide motifs to bind to the N-terminal domain of ClpX, thereby tethering substrate complexes to ClpXP. The combined use of a weak substrate recognition signal and a delivery factor that tethers the substrate to the protease allows regulated proteolysis of UmuD͞D in the cell. Dual recognition strategies of this type may be a relatively common feature of intracellular protein turnover.

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confidence: 99%

Distinct peptide signals in the UmuD and UmuD′ subunits of UmuD/D′ mediate tethering and substrate processing by the ClpXP protease

Neher

Sauer

Baker

2003

Proc. Natl. Acad. Sci. U.S.A.

112

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“…Neither the C17A nor the added C-terminal cysteine residue have any effect on DNA binding and protease sensitivity of Rep and Vir (supplemental Fig. S1, A-C), as previously described (9,12). The repressor protein with the C17A mutation and the added C-terminal cysteine (i.e.…”

Section: Methodsmentioning

confidence: 99%

“…Bacterial Strains, Plasmids, and Proteins-Expression vectors pHS502 (Rep), pHS504 (Vir3060), pGTN206 (VirC17A, where the single cysteine at residue 17 has been replaced with an alanine), pGTN204 (RepC197, which has an engineered C-terminal cysteine at residue 197 and the C17A alteration), and pGTN211 (VirC192; engineered C-terminal cysteine at residue 192 and the C17A alteration) were used to express the indicated proteins, which were purified as previously described (11)(12)(13). The QuikChange TM site-directed mutagenesis kit (Stratagene) was used to introduce single amino acid replacements in the repressor coding sequence.…”

Section: Methodsmentioning

confidence: 99%

Activation of a Dormant ClpX Recognition Motif of Bacteriophage Mu Repressor by Inducing High Local Flexibility

Marshall‐Batty

Nakai

2008

Journal of Biological Chemistry

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The C-terminal domain (CTD) of bacteriophage Mu immunity repressor (Rep) regulates DNA binding by the N-terminal domain and degradation by ClpXP protease. Five residues at the Rep C terminus (CTD5) can serve as a ClpX recognition motif, but it is dormant unless activated, a state that can be induced by the presence of dominant-negative mutant repressors (Vir). Conversion of Rep to ClpXP-sensitive form was associated with not only increased exposure of CTD5 to solvent but also increased CTD motion or flexibility as measured by fluorescence anisotropy. CTD mutations (V183S, K193S, and V196S) promoting ClpXP resistance without destroying the recognition motif prevented increased CTD motion induced by Vir. Suppression of ClpXP protease resistance conferred by the V196S mutation also correlated with restoration of CTD motion. The temperature-sensitive R47Q mutation present in cis within the DNA-binding domain restored ClpXP protease sensitivity to the V196S mutant, and anisotropy analysis indicated that R47Q allows the V196S CTD to gain increased flexibility when Vir was present. The results indicate that the CTD functions to turn the recognition motif on and off, most likely by modulating flexibility of the domain that harbors the ClpX recognition motif, suggesting a general mechanism by which proteins can regulate their own degradation.Bacteriophage Mu immunity repressor (Rep) 3 establishes and maintains lysogeny by binding to Mu DNA segments (O1, O2, and O3) that act as both operator (1, 2) and transposition enhancer (3, 4), shutting down transposition functions necessary for Mu replication. Certain physiological conditions, such as starvation or entry into stationary phase (S derepression), can promote degradation or inactivation of the repressor, leading to derepression of these transposition functions (5-8). The C-terminal domain (CTD) of Rep not only contains the recognition motif for initiating proteolysis but also modulates association of the N-terminal DNA-binding domain (DBD) with DNA.In the wild-type repressor (Rep), the CTD is located in close proximity to the DBD (9), a state that we refer to as the closed conformation and that may sterically inhibit interactions of the DBD with DNA. Upon DNA binding, the CTD moves away from the DBD (open conformation). At elevated temperatures, the CTD of temperature-sensitive (ts) repressors, such as the cts62 repressor, which has a R47Q mutation in the DBD, fails to move, and DNA binding is prevented (9). The CTD plays a major role in eliciting the temperature-sensitive DNA binding properties of these mutants; deletions (10) and single amino acid replacements (11) within the CTD can suppress ts mutations to restore stable binding at elevated temperatures. The interconversion of repressor between protease-sensitive and protease-resistant forms also correlates with movement of the CTD; however, the relevant property affected in this interconversion is not the proximity of the CTD to the DBD but rather features such as the exposure of the CTD to solvent (12). Neverthe...

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“…The close proximity of the CTD to the DBD is associated with low DNA binding affinity, suggesting that the CTD may sterically inhibit interactions of the DBD with DNA. Vir also induces movement of the Rep CTD such that its C terminus is exposed (18). Biochemical analysis of Rep and repressor CTD mutants has indicated the likelihood that a ClpX recognition motif, required for the recognition of Rep as a protease substrate, is present within the CTD.…”

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confidence: 99%

“…We suggest that the ClpX recognition motif of Rep may be activated when attached to disordered or flexible domains that confer ligand flexibility. (27), ClpP (28), Rep, and Vir3060 proteins (17,18) were purified as described previously. Green fluorescent protein (GFP) fusion proteins, which contain an N-terminal His 10 tag, were overproduced in strain BL21(DE3) (Novagen) from plasmid vectors described below.…”

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confidence: 99%

A Context-dependent ClpX Recognition Determinant Located at the C Terminus of Phage Mu Repressor

Defenbaugh

Nakai

2003

Journal of Biological Chemistry

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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.

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trans-Targeting of the Phage Mu Repressor Is Promoted by Conformational Changes That Expose Its ClpX Recognition Determinant

Cited by 13 publications

References 46 publications

Distinct peptide signals in the UmuD and UmuD′ subunits of UmuD/D′ mediate tethering and substrate processing by the ClpXP protease

Distinct peptide signals in the UmuD and UmuD′ subunits of UmuD/D′ mediate tethering and substrate processing by the ClpXP protease

Activation of a Dormant ClpX Recognition Motif of Bacteriophage Mu Repressor by Inducing High Local Flexibility

A Context-dependent ClpX Recognition Determinant Located at the C Terminus of Phage Mu Repressor

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