Crystal Structure of the Human FOXK1a-DNA Complex and Its Implications on the Diverse Binding Specificity of Winged Helix/Forkhead Proteins

Abstract

Interleukin enhancer binding factor (ILF) is a human transcription factor and a new member of the winged helix/forkhead family. ILF can bind to purine-rich regulatory motifs such as the human T-cell leukemia virus-long terminal region and the interleukin-2 promoter. Here we report the 2.4 Å crystal structure of two DNA binding domains of ILF (FOXK1a) binding to a 16-bp DNA duplex containing a promoter sequence. Electrophoretic mobility shift assay studies demonstrate that two ILF-DNA binding domain molecules cooperatively bind to DNA. In addition to the recognition helix recognizing the core sequences through the major groove, the structure shows that wing 1 interacts with the minor groove of DNA, and the H2-H3 loop region makes ionic bonds to the phosphate group, which permits the recognition of DNA. The structure also reveals that the presence of the C-terminal ␣-helix in place of a typical wing 2 in a member of this family alters the orientation of the C-terminal basic residues (RKRRPR) when binding to DNA outside the core sequence. These results provide a new insight into how the DNA binding specificities of winged helix/forkhead proteins may be regulated by their less conserved regions.The members of the extensive family of forkhead box (Fox) 3 proteins are transcription factors important for regulating cellular proliferation, transformation, differentiation, and longevity (1-3). They are expressed in various eukaryotic organisms with multiple domains that are specific for DNA binding, trans-activation, or trans-repression (4). The Fox proteins have a unique winged helix domain composed of ϳ100 evolutionarily well conserved amino acids essential for DNA recognition (5). Several three-dimensional structures of this DNA binding domain (DBD), including HNF-3␥ (FoxA3), Genesis (Foxd3), AFX (FOXO4), FREAC11 (FOXC2), and ILF-1 (FOXK1), have been determined using x-ray crystallography or NMR spectroscopy (6 -11). Most of these structures exhibit three ␣-helices, three ␤-strands, and two wing-like loops (12). The so-called winged helix DNA-binding motif of these proteins is named after the three-dimensional structure of the HNF-3␥-DNA complex in which two wing-like loops (wing 1 and wing 2) and helix 3 are involved in protein-DNA interactions (13).For many DNA-binding protein families, the differences in DNA binding specificity between family members are determined by the contact residues on recognition elements; the substitution of a DNA contact residue within family members leads to different binding specificity (14). However, transcription factors containing the winged helix-binding motif are exceptions (15). The DNA binding domains of Fox proteins exhibit a remarkable degree of sequence homology in their DNA binding region but a notable variability in their DNA recognition specificity (12). To date, more than 200 winged helix/forkhead proteins have been identified, but only two forkhead protein-DNA complexes have been reported (7, 13). Based on these two three-dimensional structures, it has been proposed that...

Structure/function relationships underlying regulation of FOXO transcription factors

“…Recently, four new crystal structures of forkhead DBD bound to the target DNA have been solved: FOXP2À DNA , NFATÀFOXP2ÀDNA , FOXK1aÀDNA (Tsai et al, 2006) and FOXO3aÀDNA (Tsai et al, 2007). Although the overall recognition of core sequence by helix H3 is in all four cases similar to that seen in FoxA3ÀDNA structure, the recognition patterns are different (Figures 5 and 6).…”

“…It seems that in the case of FoxD3, the formation of this C-terminal helix is induced by the binding to the target DNA (Marsden et al, 1998;Jin and Liao, 1999). On the other hand, the C terminus of FOXK1a DBD has helical fold both in the absence and presence of DNA, suggesting that the C-terminal helix can be a native part of the forkhead motif (Liu et al, 2002;Tsai et al, 2006). However, different classes of forkhead proteins recognize diverse DNA sequences adjacent to the core sequence through still not fully understood mechanism.…”

“…Forkhead domains of FOXO4 (human AFX) (Weigelt et al, 2001), FoxD3 (Genesis) (Marsden et al, 1998), FOXC2 (FREAC-11) (van Dongen et al, 2000) and FoxQ1 (HFH-1) (Sheng et al, 2002) contain an additional short 3 10 -type helix located between a-helices H2 and H3. Structures of DNA complexes of FoxD3 DBD (Genesis) (Jin and Liao, 1999), FOXK1a DBD (ILF-1) (Tsai et al, 2006), FOXP2 DBD and an apo-form of FOXK1a DBD (ILF-1) (Liu et al, 2002) show that these forkhead domains contain an additional helix located at the C terminus downstream of b-strand S3. It seems that in the case of FoxD3, the formation of this C-terminal helix is induced by the binding to the target DNA (Marsden et al, 1998;Jin and Liao, 1999).…”

“…However, different classes of forkhead proteins recognize diverse DNA sequences adjacent to the core sequence through still not fully understood mechanism. To date, six structures of forkhead domains bound to DNA have been solved: FoxA3ÀDNA (Clark et al, 1993), FoxD3ÀDNA (Jin and, FOXP2À DNA , NFATÀFOXP2ÀDNA , FOXK1aÀDNA (Tsai et al, 2006) and FOXO3aÀDNA (Tsai et al, 2007). Structure of FoxA3ÀDNA complex provided first details about interactions between forkhead DBD and the core sequence, showing that the helix H3 represents the main DNA recognition site .…”

Promoter-driven splicing regulation in fission yeast

“…The structure of the transcription factor FoxK1a was determined by Kuang-Lei Tsai and coworkers (Tsai et al, 2006). This transcription factor has important aminoacid sequence homology with Mei4 and Fkh2, especially in the DNAbinding domain (Figure 23, A).…”