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Hsc66 substrate specificity is directed toward a discrete region of the iron-sulfur cluster template protein IscU

Hoff, Kevin G. and Ta, Dennis T. and Tapley, Tim L. and Silberg, Jonathan J. and Vickery, Larry E. (2002) Hsc66 substrate specificity is directed toward a discrete region of the iron-sulfur cluster template protein IscU. Journal of Biological Chemistry, 277 (30). pp. 27353-27359. ISSN 0021-9258. https://resolver.caltech.edu/CaltechAUTHORS:HOFjbc02

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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.


Item Type:Article
Related URLs:
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https://doi.org/10.1074/jbc.M202814200DOIUNSPECIFIED
Additional Information:Copyright © 2002 by the American Society for Biochemistry and Molecular Biology. Received for publication, March 22, 2002, and in revised form, May 2, 2002. This work was supported by National Institutes of Health Grant GM54264, National Institutes of Health Training Grant GM07211 (to K.G.H.), and National Institutes of Health (NCI) Carcinogenesis Training Program Grant 5T32CA09054 (to T.L.T.). 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.
Subject Keywords:MOLECULAR CHAPERONE SYSTEM; ESCHERICHIA-COLI; SACCHAROMYCES-CEREVISIAE; PEPTIDE-BINDING; EXTINCTION COEFFICIENTS; HSP70 CHAPERONES; DNAK CHAPERONE; PHAGE DISPLAY; IDENTIFICATION; DOMAIN
Issue or Number:30
Record Number:CaltechAUTHORS:HOFjbc02
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:HOFjbc02
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:7307
Collection:CaltechAUTHORS
Deposited By: Lindsay Cleary
Deposited On:29 Jan 2007
Last Modified:02 Oct 2019 23:40

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