p53-DNA-binding core domain
Created by Ellie Balakhanlou
Introduction:
Tumor suppressor protein p53 (PDB ID: 1TUP) is encoded by TP53 gene on chromosome 17. Due to the complexity of the protein, it has not been completely crystallized, and this document will focus on the DNA- binding core domain.
The encoded protein can directly induce the expression of genes that regulate cell cycle. p53 has been named the “guardian of the genome’’ because of its ability to suppress mutations under DNA damaging conditions (7) . The sequence length of p53 is 390 amino acids, the molecular weight is 53 kD, and the isoelectric point is at pH 6.83 (5).
During cell stress, this protein is activated and binds to DNA to regulate transcription of genes. The critical event that leads to activation of p53 is the phosphorylation of N-terminal domain, which is the primary target for protein kinases that transfer stress signals. Oncoprotein, MDM2, binds to th N-terminal transcriptional activation domain of p53. In the cell, p53 binds to DNA which then stimulates another gene to produce p21 which interacts with cell division-stimulating protein (cdk2). When p21 complexes with cdk2, cell division is halted (10).
DNA damage could cause p53 to induce cell repair, apoptosis or halt cell cycle; thus, p53 mutations could lead to tumor development and cancer. Many of tumorigenic mutations cause deletions in the p53 gene leading to local unfolding (11). Mutated p53 cannot bind DNA effectively; therefore, p21 protein does not stop cell division. Cells begin to divide uncontrollably and form tumors. Studies suggest that p53 is mutated in over 50% of human tumors. If a person inherits only one copy of functional p53 gene, they are predisposed to cancer and could develop several types of tumors in adulthood (10).
Two proteins similar to p53 are p63 [PDB ID: 2RMN] and p73 [2XIP] and the three are members of the p53 protein family. A family includes domains with closely related amino acid sequence (in addition to folding similarities). There is about 65% amino acid identity between p53 and both p63 and p73, and about 85% identity between p63 and p73. Furthermore, all amino acid residues that are involved in DNA binding in p53 are completely conserved in p63 and p73. This suggests both conservation of sequence and structure in these structures. Proteins that are like p53 are modular molecules that have a conserved transcriptional activation domain, DNA-binding domain and an oligomerization domain. However, p63 and p73 have an additional C-terminal extension that is not visible in p53. (1).
Studies show that the three-dimensional structures of the domains of the family are very similar. The DNA binding domains of p53 and p63 have a very similar global fold and almost identical secondary structures. All members of the p53 family function as sequence-specific transcription factors. The biochemical propertythat distinguishes the family members is the discrete expression pattern exhibitedby the three proteins (8). p53 is only stabilized under cell stress and isexpressed at transcriptional level. On the other hand, p63 expression arehighly restricted and p73 is more widely expressed in specific areas of the brain.P63 is required for stratified epithelium maintenance, while P73 isinvolved in neurogenesis, neural survival, and inflammatory response. There are also differences in DNA binding ability and specificity. The additional C-terminal region in p63 and p73 that is not found in p53 could also lead to the differences in biological activity.
Basic Structure:
P53 is a tetramer that consists of four similar polypeptide chains, but only
three chains are sequence-unique. Each chain has three domains: an N-terminal transcription domain, a
core domain (residues 94-292) that is a DNA sequence specific binding domain, and a C-terminal tetramerization domain. As mentioned previously, only the core domain has been crystallized.
The secondary structure of p53 contains beta sheets, alpha helices, turns and random coils. Each subunit has an anti-parallel beta sheet sandwich and two alpha helices. There are also loop regions, the two most important being L2 and L3 which are involved in DNA binding. Loop 1, however, does not seem to bind to DNA. Alpha helix 1 (H1) is involved in dimer-dimer interaction while H2 is involved in DNA recognition and binding (6).
N-terminal region contains the transactivation domain(residues 1-62). The central core (residues 94-292) contains the DNA-binding domain (residues 102-292) which binds specifically to double-stranded target DNA that contains two decameric 'half-site' motifs 5'-Pu Pu Pu C (A/T) (T/A) GPy Py Py-3'). The C-terminal region includes the tetramerization domain (residues 325-356), and regulatory domain (residues 363-393). Tetramerization domain regulates the oligomerization state of p53, and the regulatory domain contains acetylation sites and binds DNA non-specifically. These domain are linked via linker proline-rich regions (71-93), (294-323), and a basic region that forms the C-terminus (356-393).
The DNA-binding domain of p53: The core domain-DNA complex has three p53 core domain molecules and one DNA duplex. The three domains have similar overall structures and the DNA binding does not result in any structural changes. Two of the core domains bind DNA. One of the two (blue) binds at the center of the DNA and makes contact with the bases and phosphate backbone. Another core domain (purple) binds DNA 11 bp away. These two core domains interact weakly with each other and form head-to-tail dimer. The third core domain (green) does not bind DNA, but makes protein-protein contact that stabilizes the packing (3).
The main structure of the core domain is an immunoglobin-like central beta sandwich of two anti-parallel beta sheets, creating the DNA-binding surface. This surface is created by two large loops. The longer, L2 loop occur between B strands S4 and S5, while the shorter L3 strand occurs between S8 and S9. The two loops are stabilized by a loop-sheet-helix motif (loop L1, beta-strands S2 and S2', the end of the extended beta-strand S10, and the C-terminal helix H2) and a zinc ion . The zinc ion is tetrahedrally coordinated by three cysteine side chains and a histidine side chain (2).
L3 is anchored to the minor groove of the target DNA via Arg-248. The conformation of Arginine varies from fully extended to folded, and the side chains contact the DNA directly or via water. L2 and L3 also contain the conserved residues of p53 (3).
DNA Binding Residues: The DNA-protein interaction can be explained in three parts: major groove, minor groove, and phosphate contacts. Residues 102-292 bind DNA with high specificity, and these interactions occur at the major and minor grooves of the DNA backbone. Major groove contacts the DNA at H2 helix and L1 loop. The hydrogen bonds in the major groove include Lys-120 with G8, Cys-277 with C9, and Arg-280 to G10. Minor groove contacts the binding site at Arg-248 from L3. Due to local compression in the minor groove, Arg-248 is tightly packed against sugar and phosphate groups of T-12 and T-14 inside the minor groove. It is assumed that Arg-248 plays an important role in DNA binding because it is the most frequently mutated residue in the human p53 (6).
Other DNA backbone contacts include: phosphate interactions of G10 with Ser-241 and Ala-276, and Arg-273 with G7. Lys-120 and Arg-283 interact with T8’s phosphate group (4).
Other Interactions: Arg-249 forms a salt bridge with Glu-171 and, more importantly, hydrogen bonds with the main-chain oxygens of Gly-245 and Met-246. Arg-377 also forms a salt bridge with Asp-352 which is essential for stabilization of the tetramerization domain.
Dimer interactions involve van der waals, hydrogen bonding, and electrostatic interactions. Van der waals interactions can be found between Pro-174 and His-175 of H1 helices (6).
Alternate Conformation: Studies show that p53 can exist in two specific tertiary conformations and there is a possibility of allosteric control. Two forms of p53 are the suppressor (wild-type) and oncogenic (mutant). These conformations can be recognized by antibodies. PAb1620 antibody is specific for ‘wild-type’ conformation and does not bind to denatured p53. It recognizes residues Arg-156, Leu-206, Arg-209, and Asn-210.On the other hand, antibody PAb240 can recognize residues 212-217 which are normally buried within the core domain. Thus, reactivity with PAb240 predicts the unfolded state of the protein (4).
Mutations: As mentioned previously, p53 has a central role in cell growth and survival as a tumor suppressor, apoptosis-inducer, and prognostic marker in cancer. Mutant p53 proteins are ineffective in DNA binding and can lead to cancer. Some of the mutations involve residues that bind DNA while others involve residues that are important for the structure and stability of the core domain. In many cases the unfolding of the DNA is extensive and could lead to inactivation of p53.
Although the DNA binding domain is the most conserved region, deletions are most frequent there. Mutations are most common in L3 loop, L2 loop, H2 alpha-helix and C-terminus portion of S10 strand. The mutation hotspots include: Arg-175 (6.1%) Gly-245 (6.0%) Arg-248 (9.6%)Arg-249 (5.6%) Arg-273(8.8%) Arg-282 (4.0%).These residues are near the protein-DNA interface. Arg-175 mutation unfold portions of the core domain. Arg-273 and Arg-248 mutations disrupt phosphate backbone linkages, thus, reducing DNA binding (6).
For example, the crystal structure of R249S mutant of the core domain, shows a non-native conformation of the protein (compared to the native conformation) due to a methionine switch. This methionine switch results in a conformational change that displaces DNA-contact residue (6).
Conclusion: p53 is an important protein for carcinogenesis. Thus, reactivation of p53-induced apoptosis has become a therapeutic goal. By studying this protein, researchers hope to find improvement in diagnostic techniques, accuracy of prognosis, and treatment of cancer. There are a number of on going strategies to target the p53 gene and hopefully in the near future p53 related cancers can be effectively treated (12).