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 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, JunW(Ph1) [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 T(m) of 53 degrees C compared to 16 degrees C for cFos/cJun or 44 degrees C for cFos/JunW. In PCA growth assays in the presence of cJun as competitor, phage-selected JunW(Ph1) conferred shorter generation times than JunW. Bacterial growth was barely detectable, using JunW(Ph1) 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.
Hagemann
Urs B
UB
Institute for Biology III, Albert-Ludwigs-University of Freiburg, Schaenzlestrass 1, D-79104 Freiburg, Germany.
Mason
Jody M
JM
Müller
Kristian M
KM
Arndt
Katja M
KM
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism; D030562:Databases, Protein; D017930:Genes, Reporter / Q000235:genetics; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010455:Peptides; D011485:Protein Binding; D011489:Protein Denaturation; D016760:Proto-Oncogene Proteins c-fos / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
D010446:Peptide Fragments; D010455:Peptides; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D004247:DNA