Selectional and Mutational Scope of Peptides Sequestering the Jun–Fos Coiled-Coil Domain

The activator protein-1 (AP-1) complex plays a crucial role in numerous pathways, and its ability to induce tumorigenesis is well documented. Thus, AP-1 represents an interesting therapeutic target. We selected peptides from phage display and compared their ability to disrupt the cFos/cJun interacti...

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Bibliographic Details
Published in:Journal of molecular biology 2008-08, Vol.381 (1), p.73-88
Main Authors: Hagemann, Urs B., Mason, Jody M., Müller, Kristian M., Arndt, Katja M.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
AP-1
Circular Dichroism
Databases, Protein
DNA - metabolism
Genes, Reporter - genetics
leucine zipper
library selection
Mutation - genetics
Peptide Fragments - chemistry
Peptide Fragments - metabolism
Peptides
Protein Binding
Protein Denaturation
protein design and engineering
protein stability and specificity
Proto-Oncogene Proteins c-fos - chemistry
Proto-Oncogene Proteins c-fos - genetics
Proto-Oncogene Proteins c-fos - metabolism
Proto-Oncogene Proteins c-jun - chemistry
Proto-Oncogene Proteins c-jun - genetics
Proto-Oncogene Proteins c-jun - metabolism
Thermodynamics
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Summary:The activator protein-1 (AP-1) complex plays a crucial role in numerous pathways, and its ability to induce tumorigenesis is well documented. Thus, AP-1 represents an interesting therapeutic target. We selected peptides from phage display and compared their ability to disrupt the cFos/cJun interaction to a previously described in vivo protein-fragment complementation assay (PCA). A cJun-based library was screened to enrich for peptides that disrupt the AP-1 complex by binding to the cFos coiled-coil domain. Interestingly, phage display identified one helix, JunWPh1 [phage-selected winning peptide (clone 1) targeting cFos], which differs in only 2 out of 10 randomized positions to JunW (PCA-selected winning peptide targeting cFos). Phage-selected peptides revealed higher affinity to cFos than wild-type cJun, harboring a Tm of 53 °C compared to 16 °C for cFos/cJun or 44 °C for cFos/JunW. In PCA growth assays in the presence of cJun as competitor, phage-selected JunWPh1 conferred shorter generation times than JunW. Bacterial growth was barely detectable, using JunWPh1 as a competitor for the wild-type cJun/cFos interaction, indicating efficient cFos removal from the dimeric wild-type complex. Importantly, all inhibitory peptides were able to interfere with DNA binding as demonstrated in gel shift assays. The selected sequences have consequently improved our ‘bZIP coiled-coil interaction prediction algorithm’ in distinguishing interacting from noninteracting coiled-coil sequences. Predicting and manipulating protein interaction will accelerate the systems biology field, and generated peptides will be valuable tools for analytical and biomedical applications.
ISSN:0022-2836
1089-8638
DOI:10.1016/j.jmb.2008.04.030