Abstract
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 ATPgammaS 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 10(6) 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) approximately 1 x 10(7) m(-1)). Similar results were obtained when ATP was substituted for ATPgammaS at 6 degrees 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 10(7) 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.
Highlights
Function primarily to unfold native proteins and deliver the unfolded proteins to the ClpP protease for degradation [7,8,9]
The ClpA Hexameric Ring Binds One SsrA Peptide—A synthetic 11-amino acid peptide (AANDENYALAA), corresponding to the SsrA degradation tag co-translationally added to stalled nascent polypeptide chains in vivo [32], was used to monitor peptide binding to ClpA by isothermal titration calorimetry (ITC) (Fig. 1)
Peptide titrations were performed at several temperatures between 4 and 28 °C; representative ITC titrations at 10 and 28 °C are shown in Fig. 1, A and B, respectively
Summary
12221–12230, 2005 Printed in U.S.A. The Molecular Chaperone, ClpA, Has a Single High Affinity Peptide Binding Site per Hexamer*. Substrate recognition by Clp chaperones is dependent on interactions with motifs composed of specific peptide sequences. 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 KA > 3 ؋ 107 M؊1. Substrate recognition is further affected by adaptor proteins that bind to the target protein and to the Clp chaperone and mediate or modulate their interaction. To address these issues, we have studied the binding of several relatively small peptides bearing motifs recognized by either ClpA or ClpX. We show that hexameric ClpA and ClpX bind only one peptide with high affinity and that peptides with different motifs bind at the same site or at overlapping sites
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