The role of weak non-covalent interactions in FOXO4 binding to the target DNA

Iva Zuskova^1,2, Petr Vacha^1,2, Ladislav Bumba³, Veronika Obsilova², and
Tomas Obsil^1,2

¹Faculty of Science, Charles University in Prague, 12843 Prague, Czech Republic
²Institute of Physiology, Academy of Science of Czech Republic, 14220 Prague, Czech Republic
³Institute of Microbiology, Academy of Science of Czech Republic, 14220 Prague, Czech Republic
iva.zuskova@seznam.cz

Forkhead transcription factors are structurally similar molecules containing approximately 110-amino-acid-long DNA-binding domain known as a forkhead domain. Protein FOXO4 is a member of subgroup "O" of forkhead transcription factors. Members of this subgroup play a key role in many biologically important processes [1]. For example, FOXO factors participate in metabolism control, cell-cycle control, apoptosis and oxidative stress resistance. The forkhead domain (DNA-binding domain) consists of three α-helices (H1, H2 and H3), three β-strands (S1, S2 and S3) and two flexible loops (called wings W1 and W2). The role of the wing W2 in FOXO binding to the target DNA is still elusive. Wing W2 probably interacts with the DNA in the region upstream of the core motif. It has been speculated that the FOXO DNA-binding affinity depends on A-T content (number of A-T pairs) in the region upstream of the core motif. In order to investigate this hypothesis, the DNA-binding domain of the FOXO4 protein was expressed and purified and its binding affinity for two dsDNA containing different number of A-T pairs in the region upstream of the core motif was determined using the steady-state fluorescence anisotropy-based method.

The crystal structure of the FOXO4:DNA complex suggested that both direct water–DNA base contacts and the unique water-network interactions contribute to FOXO-DBD binding to the DNA in a sequence specific manner [2]. To assess the importance of these interactions in the stability of FOXO4:DNA complex, we prepared a series of FOXO4 mutants and studied their DNA binding affinities using surface plasmon resonance.

1. K. Lin, J. B. Dorman, A. Rodan, C. Kenyon, Science 278, (1997), 1319–1322.

2. E. Boura, L. Rezabkova, J. Brynda, V. Obsilova, T. Obsil, Acta Cryst. D66, (2010), 1351–1357.

This work was funded by Grant IAA501110801 of the Grant Agency of the Academy of Sciences of the Czech Republic, by Research Project MSM0021620857 and by Research Project AV0Z50110509 of the Academy of Sciences of the Czech Republic.