(adapted from Zheng et al., 1999).
The single letter amino acid code is used for residues of E2F-4 and DP-2. Residues labelled in red are found within the RRXYD motif (see text). Bases of the E2F DNA binding site (in adenovirus E2 promoter) are labelled in blue while green circles connected by black lines represent the DNA’s sugar-phospate backbone.
HNF-3y (Clark et a l, 1993). Furthermore, there are some differences between the structures of E2F-4 and DP-2, although their individual conformations are largely the same. E2F-4 has an amino-terminal helical extension (otN) (figure 17B) that is conserved in the E2F family but not in the DP family. This feature is important in DNA sequence recognition, as we shall see later. In addition, each of the a2 and a3 helices of DP-2 are longer than the corresponding motifs in E2F-4 (figure 17B) by approximately two turns.
Protein-DNA Interactions
The phosphodiester backbone of the DNA is contacted by the amino terminus of each protein monomer’s a l helix and by segments of its p sheet (figure 17B). The a3 helix of each protein partner binds in the major groove of the DNA and participates in critical interactions with the edges of the bases (figures 17B and 17C). The contacts made by residues comprising the a3 and oN helices with the DNA are discussed below.
Recognition of the T-rich segment of the DNA-binding site (TTTCGCGCG) is related to the aN helical extension that is exclusive to the E2F partner (figure 17B). aN incorporates an invariant arginine residue (R17 in E2F-4) whose deletion abrogates DNA binding (Jordan et al, 1994). The side chain of R17-E2F penetrates deep inside the DNA’s minor groove, close to its T-rich segment. This side chain makes contacts with the 0 2 group of a thymidine base (TTTCGCGCG), as well as with the 02 and sugar groups of the neighbouring
E2F-4 and DP-2 interact in a fundamentally symmetric manner with the palindromic portion of the DNA binding site (i.e. CGCGCG), both proteins making very similar contacts to the bases. Within this portion of DNA, all of the protein-base and most of the protein-phosphate contacts are made by residues conserved in the E2F and DP families. The individual a3 helices of both proteins incorporate a conserved RRXYD motif through which E2F-4 and DP-2 each contact half of the palindromic DNA segment (i.e. CGCGCG and CGCGCG) (figure 17C). Each of the arginine residues within the motif (R56-E2F, R57-E2F and R121-DP, R 122-DP) makes two hydrogen bonds with a guanine. The individual guanine bases contacted in this manner are located in adjacent base pairs and are on opposite strands (CGC) (figure 17C). From the crystal structure, it is conceivable that the arginine residue (RRXYD) contacting the central base of the half-site (CGC), could interact with a guanine located on either strand of the DNA at this position. This reflects reports showing that some E2F DNA binding sites incorporate a cytosine base at this point in the sequence (i.e. c/gCC instead of c/gGC) (Slansky and Famham, 1996).
In the crystal structure, the contacts made by the motif’s arginine residues to the guanine bases appear to be stabilised by interactions between amino acids within the motif. The aspartic acid residue (RRXYD, i.e. D60-E2F and D 125-DP) makes a hydrogen bond with each of the motif’s arginine residues. In DP-2, a residue outside the motif also contributes to the stabilisation of these arginine residues. The side-chain of N il 8-DP forms a bridge between the DNA’s phosphodiester backbone and the guanidinium group of R 122-DP (RRXYD) (figure 17C).
The tyrosine residue in the motif (RRXYD, i.e. Y59-E2F and Y 124-DP) interacts with one of the cytosine bases in the half-site (CGC) (figure 17C). The residue makes several van der Waals contacts with the C5 and C6 atoms of the base, by virtue of its phenyl group. These close contacts reflect the preference for a cytosine or guanine base at this position in the consensus sequence (c/gGC). The tyrosine side chain’s hydroxyl group makes contacts with phosphate and sugar groups of the DNA.
Protein-protein Interactions
An extensive interface exists between E2F-4 and DP-2 that is mainly hydrophobic, involving the a l and a3 helices of each protein (figure 17D). a3- E2F packs between al-D P and a3-DP, while a3-DP packs with the interface’s two E2F helices in a reciprocal manner. The arrangement of the helices of both proteins thus confers an approximate two-fold symmetry upon the interface. This underlies observations that the protein partners can also homodimerise (section 1.4.1). Since residues in this region are highly conserved within the individual protein families, one would expect other combinations of E2F and DP family members to incorporate similar interfaces.
The imprecise symmetry at the interface is attributable to variations in the reciprocal intermolecular associations between the E2F and DP helices. In this context, differences in contact density are of particular significance. a3-E2F and a l-D P associate through 70 van der Waals contacts. In contrast, only 20 corresponding van der Waals interactions exist between a3-DP and al-E 2F.
m
FIGURE 17D