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J. Biol. Chem., Vol. 280, Issue 13, 12221-12230, April 1, 2005

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The Molecular Chaperone, ClpA, Has a Single High Affinity Peptide Binding Site per Hexamer^*

Grzegorz Piszczek, Jan Rozycki¶||, Satyendra K. Singh¶, Ann Ginsburg, and Michael R. Maurizi¶

From the Laboratory of Biochemistry, NHLBI, and the ^¶Laboratory of Cell Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892

Substrate recognition by Clp chaperones is dependent on interactions with motifs composed of specific peptide sequences. We studied the binding of short motif-bearing peptides to ClpA, the chaperone component of the ATP-dependent ClpAP protease of Escherichia coli in the presence of ATPS and Mg²⁺ at pH 7.5. Binding was measured by isothermal titration calorimetry (ITC) using the peptide, AANDENYALAA, which corresponds to the SsrA degradation motif found at the C terminus of abnormal nascent polypeptides in vivo. One SsrA peptide was bound per hexamer of ClpA with an association constant (K_A) of 5 x 10⁶ M^–1. Binding was also assayed by changes in fluorescence of an N-terminal dansylated SsrA peptide, which bound with the same stoichiometry of one per ClpA hexamer (K_A 1 x 10⁷ M^–1). Similar results were obtained when ATP was substituted for ATPS at 6 °C. Two additional peptides, derived from the phage P1 RepA protein and the E. coli HemA protein, which bear different substrate motifs, were competitive inhibitors of SsrA binding and bound to ClpA hexamers with K_A' > 3 x 10⁷ M^–1. DNS-SsrA bound with only slightly reduced affinity to deletion mutants of ClpA missing either the N-terminal domain or the C-terminal nucleotide-binding domain, indicating that the binding site for SsrA lies within the N-terminal nucleotide-binding domain. Because only one protein at a time can be unfolded and translocated by ClpA hexamers, restricting the number of peptides initially bound should avoid nonproductive binding of substrates and aggregation of partially processed proteins.

Received for publication, October 15, 2004 , and in revised form, December 28, 2004.

^* 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.

^|| Present address: Jozefa 9/37, 31-056 Krakow, Poland.

To whom correspondence should be addressed: Laboratory of Biochemistry, NHLBI, National Institutes of Health, 50 South Dr., Bldg. 50, Rm. 2341, Bethesda, MD 20892-8012. Tel.: 301-435-8082; Fax: 301-480-5492; E-mail: grzegorz_piszczek{at}nih.gov.

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