Structure determination of the closed dimeric conformation of TAp63α and investigation of its CK1 dependent activation

• Applicant: Professor Dr. Volker Dötsch
• Subject Area: Structural Biology ; Biochemistry
• Term: from 2018 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 417339402

The special role of germ cells as the source for both somatic and germ cells of all following generations requires special quality control mechanisms. Oocytes are only generated during embryogenesis and female mammals are born with a finite number of oocytes that are arrested in prophase of meiosis I. This dictyate arrest starts after passing a check point that eliminates all oocytes that have not managed to repair the DNA double strand breaks that were introduced during the preceding process of homologous recombination. This checkpoint stays active in oocytes and is responsible for the elimination of oocytes suffering from DNA damage caused for example by chemotherapeutics or irradiation as part of a cancer therapy. Consequently, female cancer patients often become infertile and suffer from premature induction of menopause. Central player of this oocyte specific quality control system is the p53-homolog TAp63 alpha. In previous research we could show that TAp63 alpha adopts an autoinhibited, compact and only dimeric conformation in resting primary oocytes. Detection of DNA damage triggers a kinase cascade that results through phosphorylation by Chk2 and CK1 in the disruption of the autoinhibited structure and the formation of open and active tetramers that induce a pro-apoptotic transcriptional program. We have optimized a bacterial expression system for the production of the autoinhibited dimer. Based on mutational analysis and SAXS Measurements we have created a first model of the autoinhibited state and have shown that the C-terminal inhibitory domain together with the N-terminal transactivation domain forms a six-stranded β-sheet that blocks the tetramerization interface. We now want to determine the high resolution structure of TAp63α using cryo electronmicroscopy. Preliminary images of TAp63 alpha in negative stain mode show images of good quality suggesting that determining the structure by cryo EM is feasible. In addition we want to investigate the mechanism of phosphorylation dependent activation. So far we have shown that activation requires phosphorylation both by Chk2 and by CK1. Phosphorylation of a single serine residue in a loop preceding the C-terminal inhibitory domain by Chk2 recruits CK1 which adds four more phosphate groups. Electrostatic repulsion leads to the disruption of the inhibitory six-stranded beta-sheet. We want to determine the phosphorylation and activation kinetics using NMR spectroscopy and identify if CK1 uses a processive or distributive mode of phosphorylation. We have also started to investigate the interaction of p63 peptides with the kinase CK1 by determining a structure of CK1 in complex with a triple phosphorylated peptide and want to further investigate the interaction of differently phosphorylated peptides by x-ray crystallography and biophysical methods.

• DFG Programme: Research Grants