Interactions with DNA

FROM MONOMER TO TETRAMER

Analytical centrifugation and labeled oligonucleotide experiments allowed researchers to understand how p73 binds to DNA. Two p73 monomers dimerize, and then two p73 dimers dimerize to form a tetramer (see Figure 3) 

1. In the absence of DNA, p73 DNA binding domain (DBD) residues are disordered
2. In presence of DNA these residues shift slightly and the DBD binds a specifically recognized sequence called a “half-site”. Two p73 proteins’ DBDs simultaneously bind to a same half-site. The two DBD monomers bind at the dimerization interface to form a dimer. Similarly, a dimer will form on an adjacent half site, providing a second dimer in close proximity to the first one.
3. The two dimers bind at the tetramerization interface, forming the final p73 tetramer 

Figure 3: p73 tetramer assembly, from a monomer to a tetramer

P73 RESPONSE ELEMENTS

All members of the p53 protein family recognize and bind to similarly organized response elements.

These response elements are made up of two successive identical half-sites, which can be separated by a spacer sequence (see Figure 4).  Each half-site usually includes 10-12 base pairs, and the spacer sequence can be 0, 1, 2 or 4 base pairs long.

Figure 4: General structure of response element recognised by p53 protein family

Every p73 DBD monomer recognizes a 5’-Pur1-Pur2-Pur3-Cyt4-Ade5/Thy5-Ade6/Thy6-Gua7-Pyr8-Pyr9-Pyr10-3’ consensus sequence which constitutes a half-site.

Once the p73 protein recognizes this sequence, its DBD binds to the quarter-site, made up of 5 base pair inverted repeats.  Two monomers each bind one quarter-site and dimerize. Two dimers that formed on successive half-sites dimerize and form tetramers whose conformations depend on the spacer sequence.

KEY RESIDUES

The DBD of p73 interacts with DNA in two manners:

1. With the bases
2. With the backbone

A loop-sheet-helix motif, namely L1-S10-H2, contains residues involved in interactions with both the bases and the backbone. Residues of the L3 loop also interact with the backbone.

1.

Interactions with the bases are via the major groove. Three key residues of the DBD interact in this manner: - Arg300

                   - Cys297

                   - Lys138

These interactions are displayed in Figure 5.

Figure 5: Arg300 (green), Cys297 (red) and Lys138 (cyan) are the key residues of the p73 DNA binding domain (orange) which interact with the nitrogenous bases of DNA (blue)

Gua4’ complementary base pairs with Cyt4 of the response element’s consensus sequence (see "P53 RESPONSE ELEMENTS") that the DBD recognises. Because the guanidinium group of Arg300 always hydrogen bonds with O6 and N7 of Gua4’ (see Figure 6), Cyt4 is the most conserved base of the half site consensus sequence.

On the other hand, there is purine degeneracy at the second and third bases of the consensus sequence, meaning that guanine or adenine can exist at either position.

This is because:

• Cys297 can be either an H-bond donor or an acceptor. It can accept an H-bond from the N4 of Cyt3’ (see Figure 6) or it can donate an H-bond to the O4 of Thy3’
• Lys138 can be promiscuous in its formation of H-bonds. It will always H-bond with the N7 of Gua2, but will sometimes simultaneously form one or more H-bonds with nearby bases. One example is its formation of an H-bond with the O6 of Gua3 (see Figure 6)

There is no direct hydrogen bonding between residues of the DBD and positions 1 and 5 of the consensus sequence.

Figure 6: Common hydrogen bonds formed between three key residues of p73 DBD and DNA bases; Arg300 always bonds with O6 and N7 of Gua4’; Cys297 can bond with N4 of Cyt3’; Lys138 always bonds with N7 of Gua2, but can also hydrogen bond with O6 of Gua3 (note: only three key residues of protein are visible- remainder of protein has been hidden for clarity)

2.

Interactions with the backbone are with the phosphate groups. Five key residues participate in this way:        -Lys138  

                     -Ala296

                     -Ser261

                     -Arg268

                     -Arg293

The latter three approach the DNA duplex from the minor groove. These protein-DNA interactions are shown in Figure 7.

Figure 7: Hydrogen bonds formed between five key residues of p73 and the phosphates of the DNA sugar-phosphate backbone 

DNA-DEPENDENT CONFORMATIONS

Depending on the length of the spacer, the DBD tetramer takes different conformations. Three of these are shown in Figure 8. See here to learn why this occurs.

Figure 8: Changes in conformation of tetramer as length of RE spacer increases from A) 0bp, to B) 2bp and C) 4bp

Techniques

• Human DBDs were expressed in E.coli and purified
• 5’ fluorescein tagged dsDNA sequences were produced as quarter, half and full sites to provide DNA binding sequences and observe different steps of p73 oligomerization
• Analytical centrifugation was used to separate and analyze the different oligomers on basis of their different sedimentation coefficients