TY  - JOUR
A1  - Strubel, Alexander
A1  - Münick, Philipp
A1  - Chaikuad, Apirat
A1  - Dreier, Birgit
A1  - Schaefer, Jonas
A1  - Gebel, Jakob
A1  - Osterburg, Christian
A1  - Tuppi, Marcel
A1  - Schäfer, Birgit
A1  - Knapp, Stefan
A1  - Plückthun, Andreas
A1  - Dötsch, Volker
T1  - Designed Ankyrin Repeat Proteins as a tool box for analyzing p63
T2  - Cell death and differentiation
N2  - The function of the p53 transcription factor family is dependent on several folded domains. In addition to a DNA-binding domain, members of this family contain an oligomerization domain. p63 and p73 also contain a C-terminal Sterile α-motif domain. Inhibition of most transcription factors is difficult as most of them lack deep pockets that can be targeted by small organic molecules. Genetic knock-out procedures are powerful in identifying the overall function of a protein, but they do not easily allow one to investigate roles of individual domains. Here we describe the characterization of Designed Ankyrin Repeat Proteins (DARPins) that were selected as tight binders against all folded domains of p63. We determine binding affinities as well as specificities within the p53 protein family and show that DARPins can be used as intracellular inhibitors for the modulation of transcriptional activity. By selectively inhibiting DNA binding of the ΔNp63α isoform that competes with p53 for the same promoter sites, we show that p53 can be reactivated. We further show that inhibiting the DNA binding activity stabilizes p63, thus providing evidence for a transcriptionally regulated negative feedback loop. Furthermore, the ability of DARPins to bind to the DNA-binding domain and the Sterile α-motif domain within the dimeric-only and DNA-binding incompetent conformation of TAp63α suggests a high structural plasticity within this special conformation. In addition, the developed DARPins can also be used to specifically detect p63 in cell culture and in primary tissue and thus constitute a very versatile research tool for studying the function of p63.
KW  - Biophysical chemistry
KW  - X-ray crystallography
Y1  - 2022
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/63272
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-632725
SN  - 1476-5403
N1  - The research was funded by the DFG (DO 545/13-1 and DO 545/18-1), the Centre for Biomolecular Magnetic Resonance (BMRZ), the Clusterproject ENABLE (funded by the Hessian Ministry for Science and the Arts) and the Clusterproject PROXIDRUGS (funded by the Federal Ministry of Education and Research).
N1  - Open Access funding enabled and organized by Projekt DEAL.
N1  - All data are fully available upon request. PDB accession codes for the four crystal structures are 7Z71, 7Z72, 7Z73, and 7Z7E.
VL  - 29
SP  - 2445
EP  - 2458
PB  - Nature Publishing Group
CY  - Houndmills, Basingstoke
ER  -