NMR structure and functional study of the DNA-binding domain of Interleukin

enhancer binding factor, a new fold in winged-helix family


Woei-Jer Chuang (莊偉哲), Pei-Phen Liu,Yu-Huei Hsieh, and Shu-Wan Chen

Department of Biochemistry, National Cheng Kung University Medical College, Tainan, Taiwan 701


Interleukin enhancer binding factors (ILFs) are transcription factors and bind to purine-rich regulatory motifs in both human T-cell leukemia virus long terminal region (HILV-1 LTR) and the interleukin-2 (IL-2) promoter. Specifically, ILFs bind to the antigen receptor response element 2 (ARRE-2) of IL-2 promoter. Although the function of ILFs is not well understood, previous studies suggest that ILFs may have a positive role in regulating IL-2 gene expression . To date, three ILFs have been found: ILF-1, ILF-2, and ILF-3 with 655, 609, and 323 amino acids, respectively. Both ILF-1 and ILF-2 contain several amino acid homologies including a region for potential ubiquitin-mediated degradation, a nuclear localization signal, an N-glycosylation motif, and a DNA-binding domain. The DNA-binding domain belongs to the winged helix family since residues from 251 to 348 of ILF-1 and ILF-2 share 35 to 88% similarity with other known members of this family. We expressed the DNA-binding domain of ILF and purified them to be homogeneous. Using gel retardation assay, we found that the C-terminal region of ILF may play an important role to regulate its binding. In addition, we determined the solution structure of the DNA-binding domain of ILF determined by heteronuclear multidimensional NMR spectroscopy and the hybrid distance geometry-dynamical simulated annealing method.  Our structural analysis revealed that the wing 2 region contains an extra a helix which may be responsible the DNA-binding specificity differences among winged helix family. This is first study to report the presence of a helix in the wing 2 region of winged helix family.