Detailed kinetic analysis of the interaction between the FOXO4–DNA-binding domain and DNA Original Research Article

The FOXO forkhead transcription factors are potent transcriptional activators involved in a wide range of key biological processes. In this work, the real-time kinetics of the interaction between the FOXO4–DNA binding domain (FOXO4–DBD) and the DNA was studied by using surface plasmon resonance (SPR). SPR analysis revealed that the interaction between FOXO4–DBD and the double stranded DNA containing either the insulin-responsive or the Daf-16 family member-binding element is preferably described by using a conformational change model which suggests a structural change of FOXO4–DBD upon binding to the DNA. This was further confirmed by using the time-resolved tryptophan fluorescence anisotropy decay measurements which revealed profound reduction of segmental dynamics of FOXO4–DBD upon the complex formation. Alanine scanning of amino acid residues engaged in polar contacts with the DNA showed that certain non-specific contacts with the DNA backbone are very important for both the binding affinity and the binding specificity of FOXO4–DBD.