Hsc66 substrate specificity is directed toward a discrete region of the iron-sulfur cluster template protein IscU.
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.
Hoff
Kevin G
KG
Department of Physiology and Biophysics, University of California, Irvine, CA 92697, USA.
Ta
Dennis T
DT
Tapley
Tim L
TL
Silberg
Jonathan J
JJ
Vickery
Larry E
LE
D000595:Amino Acid Sequence; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D007506:Iron-Sulfur Proteins; D019151:Peptide Library; D010455:Peptides; C085650:hscA protein, E coli