Base pair composition is known to play crucial roles in structure and stability properties. A/T rich segments such as a TATA box are more flexible/bendable than others 39–41
. How the change of sequence affects the DNA and the DNA-protein complex structures depends on the content of the complex. The sequence-dependent DNA deformation has been systematically studied with molecular modeling and compared with experimental data 42
. Sarai and his colleagues have shown that the AT dimer has the highest stability while the PyPu dimers such as TA present large fluctuations, depending on the flanking sequences. In our simulations, the dissociation of A/T base pairs was also observed more frequently in the Ho_CTAG complex than in others. A recent study has shown that the flexibility and bending extent of the TATA box is dependent on the context of nearby sequences, classified into flexible and rigid groups 43
. Even the concentration and type of ions present in the environment can have large impact on DNA conformations 44
. From our simulations, with everything else equal, the CATG containing DNA segment seems to be more stable and could maintain its helical conformation even in the bent state. While the more rigid CTAG containing DNA was less tolerable to conformational change and easy to suffer from base pairing disruption. It should be noted that there were other A/T base pairs in the p53-response elements. However, only the segments at the middle of the half sites were not in extensive contact with the p53 protein and therefore can undergo conformational changes.
Among the p53 response elements, the majority of the 20-base pair segments contain CATG at the centers of the half sites; however, there are also combinations of CATG, CAAG, CTTG or CTAG at the same positions 33
. Nature seems to have used the variations of the DNA sequence to modulate the response to the environmental stress. This was illustrated by previous experimental and simulation results which showed that CATG and CAAG containing p53 response elements can bind p53 efficiently and bend significantly 45,19,28
. Many factors may contribute to the DNA bending; other parts of the protein or base pairs flanking the p53-response elements may also be involved in triggering additional conformational change and in stabilizing the bent DNA conformation. There is evidence indicating that efficient p53 binding requires both the core domain and the C-terminal domain of the p53 protein 46,47
. In addition, base pairs other than the central 4 could also contribute to different extents. However, current experimental data suggest that the bending of the DNA induced by specific p53-binding is correlated with the presence of the CATG motif at the center of the half sites 45
. Balagurumoorthy et al. further observed that the CATG region in the p53 response elements is kinked; the less flexible CAAG/CTTG containing DNA segments are less so 8
; and the CTAG containing DNA segments were not observed to bend. It seems that in most cases nature has chosen more flexible sequences to allow the DNA bending required for efficient p53 binding, as was found in the majority of the p53-response elements 33
. Although other sequences such as CTAG also exist, comparative study of the p53 response elements shows that most high-affinity segments contain CATG, while the CTAG containing response elements showed binding affinities at the lower end of the spectrum 17
To further illustrate the correlation between DNA bending/efficient binding and the transactivational function, Inga et al. measured the capacity of various response elements for transactivation 48
. They found that there is a good correlation between the binding affinity of p53 and its transactivation capacity. They attempted to elucidate the relationship between features of the DNA sequence and the transactivation capacity using several parameters, such as the number of non-consensus base pairs or the location of the non-consensus pairs or both. However, no obvious trend was detected. In the end, they proposed that the transactivation capacity is correlated with the conformational change of the DNA and p53 upon p53 tetramer-DNA association. Their hypothesis was strongly supported by the measurements of these parameters for sequences that are different only in the central 4 base pairs.
While DNA bending for the 20 contiguous base pair binding site appears to be the key for the differential binding affinity, the functions of the low affinity p53 response elements and those with base-pair insertions are largely unknown. Even those with only a couple of base-pair insertions will greatly alter the p53 core domain dimer-dimer interactions and are likely to be very different from those without base-pair insertions. Studies of the structural properties could present significant difficulties for structural biologists. However, with the accumulating experimental structural results 19,20,49
and with more advanced computational power, computational studies can play a more important role in revealing the relationship between the complex structure and transcriptional selection and activity.
In summary, we have performed MD simulations on p53-DNA complexes with different DNA sequences containing CATG, CAAG, CTAG and CTTG at the centers of the half sites. Our results show that the extent of DNA bending depends on the ability of DNA to maintain its specific interactions with p53 while the p53 dimers approach each other to maximize the p53-p53 interactions. Complexes with DNA containing the CATG motif can better retain the specific interactions than the complexes with DNA containing the CTAG motif, which is associated with the intrinsic bendability of the CATG motif or the rigidity of the CTAG segment of the DNA.