Abstract
Hsc66 and Hsc20 comprise a specialized chaperone system important for the assembly of iron-sulfur clusters in Escherchia coli. Only a single substrate, the Fe/S template protein IscU, has been identified for the Hsc66/Hsc20 system, but the mechanism by which Hsc66 selectively binds IscU is unknown. We have investigated Hsc66 substrate specificity using phage display and a peptide array of IscU. Screening of a heptameric peptide phage display library revealed that Hsc66 prefers peptides with a centrally located Pro-Pro motif. Using a cellulose-bound peptide array of IscU we determined that Hsc66 interacts specifically with a region (residues 99-103, LPPVK) that is invariant among all IscU family members. A synthetic peptide (ELPPVKIHC) corresponding to IscU residues 98-106 behaves in a similar manner to native IscU, stimulating the ATPase activity of Hsc66 with similar affinity as IscU, preventing Hsc66 suppression of bovine rhodanese aggregation, and interacting with the peptide-binding domain of Hsc66. Unlike native IscU, however, the synthetic peptide is not bound by Hsc20 and does not synergistically stimulate Hsc66 ATPase activity with Hsc20. Our results indicate that Hsc66 and Hsc20 recognize distinct regions of IscU and further suggest that Hsc66 will not bind LPPVK motifs with high affinity in vivo unless they are in the context of native IscU and can be directed to Hsc66 by Hsc20.
Highlights
Hsc66 (HscA) is a 66-kDa heat shock cognate protein in Escherichia coli that belongs to the Hsp70 class of molecular chaperones [1, 2]
We have investigated Hsc66 substrate specificity using phage display and a peptide array of IscU
Peptide array, and structural studies reveal that DnaK prefers unfolded peptides approximately eight amino acids in length and enriched by hydrophobic residues flanked at either end by basic residues (16 –18)
Summary
Hsc (HscA) is a 66-kDa heat shock cognate protein in Escherichia coli that belongs to the Hsp class of molecular chaperones [1, 2]. To determine whether there is a correlation between peptides selected in the phage library and Hsc recognition of IscU, as well as to determine whether Hsc binds IscU in a specific location or at various sites, we screened a cellulose array displaying the entire sequence of IscU as overlapping peptides 13 amino acids in length. Peptides identified by both approaches have been synthesized and their effects on Hsc ATPase activity have been examined. We have found that Hsc prefers peptides with centrally located proline residues and, consistent with this finding, Hsc binds to one region of IscU that displays the sequence LPPVK
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