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. 1999 Mar 15;13(6):666-74.
doi: 10.1101/gad.13.6.666.

Structural basis of DNA recognition by the heterodimeric cell cycle transcription factor E2F-DP

N Zheng  1 , E Fraenkel, C O Pabo, N P Pavletich
Affiliations

Structural basis of DNA recognition by the heterodimeric cell cycle transcription factor E2F-DP

N Zheng et al. Genes Dev. .

Abstract

The E2F and DP protein families form heterodimeric transcription factors that play a central role in the expression of cell cycle-regulated genes. The crystal structure of an E2F4-DP2-DNA complex shows that the DNA-binding domains of the E2F and DP proteins both have a fold related to the winged-helix DNA-binding motif. Recognition of the central c/gGCGCg/c sequence of the consensus DNA-binding site is symmetric, and amino acids that contact these bases are conserved among all known E2F and DP proteins. The asymmetry in the extended binding site TTTc/gGCGCc/g is associated with an amino-terminal extension of E2F4, in which an arginine binds in the minor groove near the TTT stretch. This arginine is invariant among E2Fs but not present in DPs. E2F4 and DP2 interact through an extensive protein-protein interface, and structural features of this interface suggest it contributes to the preference for heterodimers over homodimers in DNA binding.

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Figures

Figure 1
Figure 1
The DNA-binding domains of E2Fs and DPs have limited across-family homology but share the same fold. (A) Sequence alignment of known E2F and DP family members with the E2F4 and DP2 polypeptides used in the crystallization. Residues conserved throughout the E2F and DP families are highlighted in yellow. The RRXYD DNA recognition motif is underlined in the E2F4 and DP2 sequences. (B) Sequence of the DNA duplex used in the cocrystallization, with the E2F site at the adenovirus E2 promoter underlined.
Figure 2
Figure 2
Structure of the E2F4-DP2 heterodimer DNA complex. (A) Schematic view looking down the approximate axis of twofold pseudo symmetry in the heterodimer. The DNA axis is vertical in this view. (B) View of the complex looking down the DNA axis. Figures were prepared with the programs MOLSCRIPT (Kraulis 1991) and RASTER3D (Merritt and Bacon 1997).
Figure 3
Figure 3
The E2F4 and DP2 DNA-binding domains consist of the winged-helix motif. (A) Superposition of the E2F4 and DP2 winged-helix DNA-binding domains. (B) The winged-helix domain of the HNF-3γ transcription factor (Clark et al. 1993) in an orientation obtained by aligning it with the DP2 winged-helix domain in A.
Figure 4
Figure 4
Recognition of the core DNA site by the E2F4–DP2 heterodimer is overall symmetric. (A) Figure shows the E2F4 and DP2 residues that contact the DNA bases; for clarity, only part of the α3 helices of each subunit and the amino-terminal helix of E2F4 are shown. (B) Sketch summarizes the DNA contacts made by the E2F4–DP2 heterodimer. (C) Close-up view of the interactions between the two arginines of the RRXXD motif and the two neighboring guanines within the half site.
Figure 5
Figure 5
The interface of the E2F4–DP2 heterodimer involves the α1 and α3 helices of both proteins. (A) The interface has an approximate twofold symmetry, with the axis of symmetry (indicated) perpendicular to the plane of the figure. There are many differences in the intermolecular contacts, with the DP2 α1 and E2F4 α3 helices packing more extensively than the reciprocal E2F4 α1 and DP2 α3 helices. The intermolecular contact density was calculated by considering contacts made by each amino acid with an interatomic distance <4 Å. (B) Stereo view of the E2F4–DP2 interface. To make the interface easier to see, only residues that make multiple van der Waals contacts (< 4.0 Å) are illustrated. A salt bridge is formed between the α1 helix of DP2 and α3 helix of E2F, contributing to the asymmetry at the interface.
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