(Zheng et al., 1999).
The interface is formed by the a l and a3 helices of both proteins. In the interests of clarity, only those residues are shown that participate in making multiple van der Waals contacts.
Among the residues at the densely-packed half of the interface, for example, are V64-E2F (a3) and L71-DP ( a l) (figure 17D). Their side chains contact each other, as well as additional interface residues, through a total of 21 intermolecular interactions. However, the number of contacts made by the corresponding residues in a3-DP and al-E 2F, in the sparsely-packed half of the interface, is significantly lower. VI29-DP (a3) is involved in six intermolecular interactions and L22-E2F ( a l) in only one.
The asymmetry discussed above arises from the distinct inter-helical distances within each protein. The distance between al-D P and a3-DP is about 3 Â shorter than that between the equivalent E2F helices. This is due to the relative size of the hydrophobic residues in the core of each protein. Small hydrophobic residues, for example V78-DP and A 126-DP, interact with each other at the a l- a 3 interface of DP-2. The analogous helices in E2F-4, however, pack against each other (in general) through larger residues. For example, F29-E2F and I61-E2F correspond to V78-DP and A 126-DP respectively. Residues at the a l- a 3 interface within the individual proteins are conserved in their respective families. The structures of other E2F and DP family members therefore, will probably differ in a manner comparable to that discussed here. One can also conclude, from the difference in inter-helical distances described above, that the protein-protein interfaces within the homodimers would be distinct from that within a heterodimer.
A comparison of the interface residues belonging to the monomers reveals that approximately two thirds of these amino acids differ between E2F-4 and DP-2. These differences also confer asymmetry upon the interactions at the interface. At
the densely-packed half of the interface for example, a salt bridge is formed between E66-E2F and R72-DP (figure 17D), both of which are conserved in their respective families. In contrast, the corresponding residues (G72-E2F and M131- DP) in the sparsely-packed interface portion do not interact. Furthermore, the salt bridge between arginine and glutamic acid described earlier, could not form between monomers of the same protein family. The asymmetric features of the interface outlined above, contribute to the preference exhibited by E2F and DP to bind DNA as heterodimers.
Implications for Aptamers of the E 2 F - l A c y c Homodimer
The structure described above shows that all of the residues of E2F-4 and DP-2 that interact with the DNA bases, as well as most of those that associate with the phosphodiester backbone, are conserved within the individual protein families. Combinations of winged helix domains from other E2F and DP family members therefore, would be expected to interact with the DNA binding site in a very similar manner. The case of the E2F-lAcyc homodimer, however, would be somewhat different. Although the SELEX experiments described in this chapter did not generate aptamers for E2F-lAcyc, I will outline here how the crystal structure might relate to DNA sequence selection by this recombinant protein.
The winged-helix domain of E2F-1 (equivalent to the domain of E2F-4 in the crystal structure) encompasses residues 112-195 (Zheng et a l, 1999). However,
E2F-lAcyc contains an additional region outside this domain, since it incorporates residues 95-195. It is possible that the additional residues partly modulate
sequence specificity and that as a result, E2F-lAcyc would interact preferentially with a slightly different sequence compared to that recognised by the E2F-4
fragment in the crystal structure. This point is even more relevant in the context of full-length E2F and DP heterodimers. The different preferences of such heterodimers for specific sequences (section 1.4.4.3) may also be attributable to residues outside the winged-helix domains. It is worth mentioning at this point that in the cellular environment, other proteins bound to the heterodimers may also play a role in modulating sequence specificity by interacting with bases outside the consensus site.
The crystallographic data shows that while each winged-helix domain contacts half of the palindromic DNA sequence (CGCGCG), the asymmetry in the extended binding-site (TTTCGCGCG) is exclusively associated with the otN helical extension of E2F-4, that is conserved in the E2F family. It is conceivable that the E2F-lAcyc homodimer, that also incorporates the (xN component, would preferentially select an entirely palindromic site, perhaps with a central core comprising C/G bases, flanked by a T-rich segment at the 5’ end and a corresponding A-rich segment at the 3’ end.
As explained above, the protein-protein interface within a homodimer would differ from that within a heterodimer, owing to the distinct interhelical arrangement associated with the winged helix-domain of each protein partner. This might generate subtle differences between the DNA sequences recognised by E2F homodimers, DP homodimers and heterodimers.
3
.4.5
SELEX and E2F - The Next Step
Although elucidation of an E2F heterodimer’s crystal structure rendered that of the homodimer somewhat redundant, and SELEX experiments have been successfully performed with E2F heterodimers, there may yet be a future role for SELEX. This pertains to the specificity of individual E2F heterodimers for distinct genes. I have already discussed the current data regarding such specificity (sections 1.4.3.3 and 1.4.4.3) However, there are caveats associated with the experimental approaches employed to obtain these results, examples of which follow.
The experiments conducted by DeGregori and colleagues (DeGregori et a l, 1997) involved the infection of quiescent REF52 cells with adenoviral vectors expressing each of the E2F proteins. Since the latter are not at physiological levels, they may bind to promoters owing to their increased concentration. The abnormally high concentration may also abolish significant protein-protein interactions. Furthermore, the overexpression of a certain E2F protein during quiescence does not necessarily reflect the normal timing of its expression in the cell cycle (Famham, 1996; Slansky and Famham, 1996; Wells et a l, 2000).
The SELEX experiments performed by Tao and colleagues demonstrate that different E2F heterodimers have distinct preferences for the precise sequences they bind (Tao et a l, 1997). However, since the evolved sequences are not those
of natural promoters, a link between a specific heterodimer and a certain promoter cannot be established.
In the context of E2F specificity, a recent study may represent an improvement in experimental design (Wells et a l, 2000). Wells and co-workers treated NIH 3T3 cells with formaldehyde in order to cross-link transcription complexes to the promoter DNA. This was followed by immunoprécipitation with antibodies against specific E2F proteins or members of the pRb family. The co-precipitating DNA was analysed by promoter-specific PCR primers allowing specific proteins and DNA sequences to be matched according to their interactions in vivo.
However, it appears that the use of different antibodies that are specific for the same protein may generate conflicting results (perhaps due to the variation in epitope accessibility) (Wells et a l, 2000).
A modified form of SELEX, known as Genomic SELEX, may also prove to be informative in these specificity studies. In this case, a genomic DNA library replaces the degenerate pool of oligonucleotides. This variation of the SELEX procedure thus allows the identification of sequences bound with highest affinity
in vivo by a protein (Gold et al, 1995). However, since the binding reaction
conditions that are chosen could affect the affinities of the individual E2F proteins for specific promoters, the results obtained from such an approach would only serve to complement studies carried out using other methods.