Structural characterization of the Forkhead box O4 (FOXO4): complex

P­04.1­01

R. MandalI, O. PetrvalskáII, K. KohoutováI, V. ObšilováII, T. ObšilI

IDepartment of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague 2, Czech Republic, IIDepartment of Structural Biology of Signaling , Division BIOCEV, Institute of Physiology of the Czech Academy of Sciences, Vestec, Czech Republic

The forkhead box O (FOXO) plays an important role in cell cycle regulation, metabolism, stress resistance and longevity of mammals. In human, FOXO family consists of four members: FOXO1, FOXO3, FOXO4 and FOXO6 which share a highly conserved DNA­binding domain called the forkhead box domain. They are expressed in all mammalian tissues and their signal is regulated by the interaction with other proteins and post­translation modification including phosphorylation, acetylation and ubiquitination. In many human cancers, FOXO4 has been considered as a tumour suppressor and involved in the interaction with several proteins including p53 to regulate apoptosis, cancer and age­related diseases. The tetrameric p53 protein is involved in the regulation of more than 500 target in the biological network. Recently, it has been reported that upon ionizing radiation (IR) damage, FOXO4 interacts with p53 which promotes over apoptosis and maintains the senescent cell viability by blocking p53 mediated apoptosis. However, the molecular details of FOXO4:p53 interaction remain unclear due to the absence of structural data. Therefore, the main aim of this project is to investigate the structural basis of the FOXO4:p53 complex. For that reason, sedimentation velocity analytical ultracentrifugation (SV­AUC), 2D 1H­15N HSQC NMR spectroscopy, chemical cross­linking coupled to mass spectroscopy and molecular docking experiments were conducted to obtain the molecular insight of the FOXO4:p53 complex with various constructs of FOXO4 and p53. Experimental data suggest that both proteins interact with the binding affinity in the micromolar (μM) range and that the N­terminal segments, as well as other regions of FOXO4­ DNA binding domain are involved in the interaction with p53. This work was supported by the Charles University Grant Agency (project: 251203).