The molecular chaperone, ClpA, has a single high affinity peptide binding site per hexamer
The molecular chaperone, ClpA, has a single high affinity peptide binding site per hexamer
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DOI：
10.1074/jbc.m411733200
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发表时间：
2005-04-01
影响因子：
4.8
通讯作者：
Maurizi, MR
中科院分区：
文献类型：
--
作者：
Piszczek, G;Rozycki, J;Maurizi, MR
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 ATP gamma S 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 similar to 1 x 10(7) M-1). Similar results were obtained when ATP was substituted for ATP gamma S 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.
