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J. Biol. Chem., Vol. 283, Issue 14, 9060-9070, April 4, 2008

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

From the Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, D. C. 20057

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.

Received for publication, July 5, 2007 , and in revised form, December 13, 2007.

^* This work was supported by National Institutes of Health Grant GM58265 (to H. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ Recipient of a predoctoral fellowship award from the National Institutes of Health (Ruth L. Kirschstein National Research Service Award GM070441).

² To whom correspondence should be addressed: Dept. of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Box 571455, 3900 Reservoir Rd. NW, Washington, D. C. 20057-1455. Tel.: 202-687-1442; Fax: 202-687-7186; E-mail: nakaih{at}georgetown.edu.

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