Fox protein 家族成員都具有約100 個胺基酸、高度保留核酸結合區域稱為Winged-Helix DNA-Binding Domain (簡稱WH-DBD)；WH-DBD的結構包含三個 α-helix、三個 β-strand 以及兩個『Wing-like』的loop。WH-DBD 有三個區域會與DNA 作用：(一) Helix 3，辨識DNA 特定的序列並與『major groove』作用；(二) Wing 1，與DNA backbone 帶負電的磷酸根作用；(三) Wing 2，與DNA 的『minor groove』作用。肌細胞核因子 (MNF) 是以一種專門表現在衛星細胞的轉錄因子；在肌細胞受傷後，MNF 會調控衛星細胞有關生長、以及分化基因的表現。兩種MNF：MNF-α與 MNF-β (分別擁有617、414 個胺基酸)，藉由選擇性接合的方式 從同一MNF 基因中分別被表現出來。這兩種蛋白都具有FHA (forkhead-associated domain)與核酸結合區域。在此研究中我們藉由核磁共振技術決定了MNF 的核酸結合區域的三維結構。與間白素結合因子(ILF) 類似，包含四個 α-helix、三個 β-strand、一個type I turn 以及一個『Wing-like』狀的loop；順序為：H1-T1-S1-H2-H3-S2-W1-S3-H4。與同族的WH-DBD 相較，間白素結合因子的WH-DBD 在C 端形成 α-helix而非典型的Wing-like loop。轉錄因子與DNA 的作用是一個結構上高度動態的過程。為了比較不同WH-DBD 之間動力學的差異，我們利用氮十五核磁共振遲緩實驗還有自由模式方程式提供有關蛋白骨架在ps/ns，μs/ms 運動的資訊。間白素結合因子 (ILF) 與肌細胞核因子 (MNF)WH-DBD 的N、C 端、H2-H3 loop 以及S2-S3 loop 都擁有較高的可動性，根據蕭傳鐙老師實驗室所解出的ILFDBD-DNA complex 的結構中可知可動性高的區域，如N 端、W1 與H2-H3 loop，也會與DNA 作用。T34、K35 (H2 末端)、R41 與G46 (H2-H3 loop) 的backbone 在ILF 與MNFDBD中都具有大程度的『Conformational Exchange (Rex)』的性質。比較另一個WH-DBD，『Genesis-DBD』的動力學參數，我們發現Genesis-DBD 在對應的區域 (34-46) 並沒有任何Rex；雖然ILFDBD 與Genesis-DBD 有53%的相似度，但是他們卻會辨識不同的DNA 序列，ILF 與MNFDBD辨識的DNA 核心序列同為T-(G/A)-TTTAC、Genesis-DBD 則主要為GTTATTTT，他們具備了不同的DNA-Binding 性質；另一方面，許多研究中認為WH-DBD H3 的相對位置，H2-H3 loop、Wing 1 與C-terminal的氨基酸組成，甚至是電荷分布的變異性是造成此家族擁有不同DNA 辨識性質的原因。根據序列的比較可知在H2-H3 loop ( 42-46 )如EKFPA 在Genesis-DBD、TADKG 在ILF 與MNF；C-terminal ( 85-93 )如AKLIEQAFR在 ILF、AKLVEQAFR 在 MNF、DMFDNGSFL 在 Genesis 都十分不同。我們推測此兩區域的動力學性質與結構是造成此家族蛋白擁有多樣DNA-Binding 性質的原因。本研究說明了winged-helix proteins 在動力學與結構上的多樣性，提供winged-helix protein 與DNA 作用新的觀點。

Members of the winged helix/forkhead family are characterized by a conserved 100-amino acid DNA-binding domain that contains three α-helices,three β-strands, and two wing-like loops. Three major regions of winged-helix/forkhead proteins are involved in protein and DNA interactions: (1) helix 3, the recognition site which makes sequence-specific contacts with the major groove of DNA; (2) wing 1, which makes phosphate contact with the backbone of DNA; and (3) wing 2, which interacts with the minor groove of DNA. MNFs are transcription factors that are selectively expressed in myogenic stem cells. MNFs regulate the genes that coordinate the proliferation and differentiation of myogenic stem cells after muscle injury.Two MNF isoforms were found: MNF-α and MNF-β contain 617 and 414 amino acids, respectively. Although they are derived from a single mnf gene by alternative splicing, the expression of MNF-α and MNF-β is differentially regulated. Both MNF-α and MNF-β contain several domains, including the forkhead-associated domain and the DNA-binding domain. In this study we have determined 3D structure the DNA-binding domain of myocyte nuclear factor (MNF) by NMR spectroscopy. Similar to 3D structure of the DNA-binding domain of interleukin enhancer binding factor (ILF), it consists of four α-helices, three β-strand, and one wing, arranged in the order H1 -S1-H2-H3-S2-W1-S3-H4. In contrast to other proteins of this family, the DNA-binding domain of MNF and ILF contains a C-terminal α helix in place of a typical wing 2. It is known that the recognition between transcription factors and their DNA binding sites is a highly dynamic process. To compare the dynamic properties of the winged-helix proteins with DNA, we used 15N NMR relaxation measurements and the model-free formalism to provide insight into protein dynamics on ps-ns and μs-ms time scales. High flexibility of backbone dynamics of the DNA-binding domain of ILF and MNF were observed for the residues in the loop between H2 and H3 and wing 1, as well as the C-terminal region. Based on 3D structure of the ILF/DNA complex determined by Dr. Chwan-Deng Hsiao’s group, the loop between H2 and H3, helix 3, wing 1, and the C-terminal basic region make specific contacts with DNA. These regions except helix 3 were dynamic according to our NMR study. Both T34 and K35 at the C-terminus of H2 and R41 and G46 of the H2-H3 loop of ILF and MNF showed high conformational exchange (Rex) on μs-ms time scale compared to those of Genesis, a winged helix protein. Although the DNA-binding domain of MNF and ILF shares 53% with that of Genesis, they recognize diverse DNA sequence. The DNA sequence T-(G/A)-TTTAC is specifically recognized by ILF and MNF. In contrast, Genesis recognizes the DNA sequence GTTATTTT. Many reports have suggested that such diverse recognition is possibly due to the relative orientation of the DNA-recognition helix, amino acid compositions of the H2-H3 loop, wing 1, and C-terminal region, as well as the electrostatic surface potentials present in winged helix/forkhead proteins. The sequence analysis showed that the amino acid sequences of the H2-H3 loop (residues 42-46 ) i. e., EKFPA in Genesis and TADKG in ILF and MNF and the C-terminal region (residue 85-93) i. e., AKLIEQAFR in ILF, AKLVEQAFR in MNF, and DMFDNGSFL in Genesis are very different. These results suggest that the dynamic properties and structure of these regions may play an important role in DNA interaction and results in its difference in DNA-binding specificity. This study demonstrates a possible structural and dynamic diversity among the winged-helix proteins and provides new insights
into the mechanism of DNA recognition in these proteins.

Aoki, M., Jiang, H. and Vogt, P.K. Proteasomal degradation of FoxO1
transcriptional regulator in cells transformed by the P3k and Akt oncoproteins.
PNAS. 14, 13613-13617 (2004)
Banham, A.H., Beasley, N., Campo, E., Fernandez, P.L., Fidler, C., Gatter, K.,
Jones, M., Mason, D. Y., Prime, J. E., Trougouboff, P., Wood, K. and Cordell,
J. L. The FOXP1 winged helix transcription factor is a novel candidate tumor
suppressor gene on chromosome 3p. Cancer Res. 61, 8820-8829 (2001)
Bravieri, R., Shiyanova, T., Chen, T. H., Overdier, D. and Liao, X. Different
DNA contact schemes are used by two winged helix proteins to recognize a
DNA binding sequence. Nucl. Acids Res. 25, 2888-2896 (1997)
Brunger, A.T. X-PLOR Version 3.1: A system for X-ray crystallography and
NMR. New Haven, CT: Yale University Press (1992)
Carlsson, P., Mahlapuu, M. Forkhead Transcription Faxtors：Key Players in
Development and Metabolism. Dev Biol. 250, 1-23 (2002)
Chuang WJ, Yeh IJ, Hsieh YH, Liu PP, Chen SW, Jeng WY. 1H, 15N and 13C
resonance assignments for the DNA-binding domain of myocyte nuclear
factor (Foxk1). J Biomol NMR. 24, 75-76 (2002)
Clark, K.L., Halay, E.D., Lai, E., Burley, S.K. Co-crystal structure of the
HNF-3/fork head DNA-recognition motif resembles histone H5. Nature. 364,
412-20 (1993)
Cordier, F., Caffrey, M., Brutscher, B., Cusanovich, M.A., Marion, D.,
Blackledge, M. Solution structure, rotational diffusion anisotropy and local
backbone dynamics of Rhodobacter capsulatus cytochrome c2. J Mol Biol. 14,
341-361 (1998)
Gajiwala, K. S. and Burley, S. K. Winged helix proteins. Curr. Opin. Struct.
Biol. 10,110-116. Review (2000)
Hawke, T.J., Jiang, N. and Garry, D.J. Absence of p21CIP rescues myogenic
progenitor cell proliferative and regenerative capacity in Foxk1 null mice. J
Biol Chem. 278, 4015-4020 (2003)
Jin, C., Marsden, I., Chen, X., Liao, X. Dynamic DNA contacts observed in
the NMR structure of winged helix protein-DNA complex. J Mol Biol. 289,
683-690 (1999)
Jin, C., Marsden, I., Chen, X., Liao, X. Sequence specific collective motions
in a winged helix DNA binding domain detected by 15N relaxation NMR.
Biochemistry. 37, 6179-6187 (1998)
Katoh, M., Katoh, M. Human FOX gene family (Review). Int J Oncol. 25,
1495-1500 (2004)
Kaufmann, E., Knochel, W. Five years on the wings of fork head. Mech Dev.
57, 3-20. Review (1996)
Kaestner, K. H., Knöchel, W. and Martínez, D. E. Unified nomenclature for
the winged helix/forkhead transcription factors. Genes Dev. 14, 142-46 (2000)
Li, C., Lai, C.F., Sigman, D.S. and Gaynor, R.B. Cloning of a cellular factor,
interleukin binding factor, that binds to NFAT-like motifs in the human
immunodeficiency virus long terminal repeat. PNAS. 88, 7739-7743 (1991)
Liu, P.P., Chen, Y.C., Li, C., Hsieh, Y.H., Chen, S.W., Chen, S.H., Jeng, W.Y.,
Chuang, W.J. Solution structure of the DNA-binding domain of interleukin
enhancer binding factor 1 (FOXK1a). Proteins 49, 543-553 (2002)
Lipari, G., & Szabo, A. Model-free approach to the interpretation of nuclear
magnetic resonance relaxation in macromolecules. 1. Theory and range of
validity. J. Am. Chem. Soc. 104, 4546–4559 (1982)
Marsden, I., Chen, Y., Jin, C., and Liao, X. Evidence That the DNA Binding
Specificity of Winged-Helix Proteins Is Mediated by a Structural Changes in
the Amino Acid Sequence Adjacent to the Principal DNA Binding Helix.
Biochemistry 36, 13248-13255 (1997)
Nirula, A., Moore, D.J., Gaynor, R.B. Constitutive binding of the
transcription factor interleukin-2 (IL-2) enhancer binding factor to the IL-2
promoter. J Biol Chem. 272, 7736-7745 (1997)
Overdier, D.G., Porcella, A., Costa, R.H. The DNA-binding specificity of the
hepatocyte nuclear factor 3/forkhead domain is influenced by amino-acid
residues adjacent to the recognition helix. Mol Cell Biol 14, 2755-2766 (1994)
Pierrou, S., Hellqvist, M., Samuelsson, L., Enerback, S., Carlsson, P. Cloning
and characterization of seven human forkhead protein: binding site specificity
and DNA bending. EMBO J. 13, 5002-5012 (1994)
Sheng, W., Rance, M., Liao, X. Structure comparison of two conserved
HNF-3/fkh proteins HFH-1 and genesis indicates the existence of folding
differences in their complexes with a DNA binding sequence. Biochemistry.
12, 3286-3293 (2002)
Shi, C., Zhang, X., Chen, Z., Sulaiman, K., Feinberg, M.W., Ballantyne, C.M.,
Jain, M.K., Simon, D.I. Integrin engagement regulates monocyte
differentiation through the forkhead transcription factor Foxp1. J Clin Invest.
114, 408-418 (2004)
Sutton, J., Costa, R., Klug, M., Field, L., Xu, D., Largaespada, D. A., Fletcher,
C. F., Jenkins, N. A., Copeland, N. G., Klemsz, M., and Hromas, R. Genesis,
a winged helix transcriptional repressor with expression restricted to
embryonic stem cells. J. Biol. Chem. 271, 23126 (1996)
Shiyanova, T. and Liao, X. The dissociation rate of a winged helix
protein-DNA complex is influenced by non-DNA contact residues. Arch.
Biochem. Biophys. 362, 356-362 (1999)
Van Dongen, M.J., Cederberg, A., Carlsson, P., Enerback, S., Wikström, M.
Solution structure and dynamics of the DNA-binding domain of the
adipocyte- transcription factor FREAC-11. J Mol Biol. 296, 351-359 (2000)
Weigelt, J., Climent, I., Dahlman-Wright, K., Wikstrom, M. Solution
structure of the DNA binding domain of the human forkhead transcription
factor AFX (FoxO4). Biochemistry. 22, 5861-5869 (2001)
Wüthrich, K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc.
(1986)
Yang, Q., Bassel-Duby, R., Williams, R.S. Transient expression of a wingedhelix
protein, MNF-beta, during myogenesis. Mol Cell Biol. 17, 5236-43
(1997)
Yan, H. and Liao, X. Amino acid substitutions in a long flexible sequence
influence thermodynamics and internal dynamic properties of winged helix
protein genesis and its DNA complex. Biophys J. 85, 3248-54 (2003)
謝玉惠，間白素結合因子 (ILF) 核酸結合區的功能及其結構之研究。國
立成功大學生物化學研究所碩士論文，1998。
劉佩棻，利用核磁共振光譜決定間白素結合因子 (ILF) 核酸結合區的水
溶液中三度空間結構。國立成功大學生物化學研究所碩士論文，2000。
葉意茹，肌細胞因子(MNF)的核酸結合區域之表現與三度空間結構。國立
成功大學生物化學研究所碩士論文，2001。
孫千惠，間白素結合因子與去氧核醣核酸結合的特性之探討。國立成功
大學生物化學研究所碩士論文，2002。
蔡光磊，間白素結合因子核酸結合區與核酸複合體之晶體結構分析與研
究。國立清華大學生物資訊與結構生物研究所碩士論文，2004。