Tumor Suppressor p53 Complexed with DNA (PDB ID: 1TUP) from Homo sapiens
Created by: Samantha Clark
p53 complexed with DNA (PBD ID: 1TUP) is a tumor suppressor in human somatic cells and one of the most commonly observed genetic factors in tumor growth. The gene coding for p53 is located on the short arm of human chromosome 17. The product protein’s isoelectric point is 8.91, consists of 3 subunits and its molecular weight is 73616.39 Da (1, 2). While wild type p53 acts as a tumor suppressor, mutations in the protein are the most commonly seen genetic root of the development of tumors. Approximately half of cancers involve mutations of one p53 allele in which the second allele is deleted, resulting in a loss of function that inactivates p53’s tumor suppression properties. Remaining cases of cancer often involve cellular or viral oncogenes that inactivate p53. Wild type p53 inhibits neoplastic growth in a number of ways, either by inhibiting the growth of tumor cells, blocking transformation by an activated oncogene, or preventing tumor formation. p53 controls neoplastic growth by regulating a cell cycle checkpoint. This checkpoint primarily deals with the genetic integrity of the cell—if p53 detects DNA damage, the protein initiates cell cycle arrest. Overexpression of p53 results in the arrest of the cell cycle in G1; thus, the pathway by which p53 initiates apoptosis is a common target in cancer-treating drugs.
Because p53’s main role in the cell consists of checking the DNA for damage before cellular replication, its structure consists largely of a DNA binding domain. The core structure of p53 has an antiparallel β-sheet sandwich with four or five β-strands. A loop-sheet-helix packs closely against one end of this β-sheet. At the same end as the loop-sheet-helix, a tetrahedrally coordinated zinc atom anchors two large loops, compensating for the lack of hydrogen bond strength in this area of p53’s core domain. The larger loop, L2 directly binds to the DNA while the smaller loop, L3 packs against the larger DNA binding loop L2 (the loop L1 is a part of the loop-sheet-helix motif). The β-sandwich is a large part of the structure of the core domain but is not involved in the binding of DNA. The core domain uses the loop-sheet-helix motif and one of the two large loops to bind DNA. The helix and the loop of the loop-sheet-helix fits into the major groove of the DNA and the large loop of p53’s structure provides an arginine residue that then binds with the minor groove of the DNA.The two large loops of p53 have little regular secondary structure. The loops lack the extensive hydrogen bond network of the rest of the protein, but their shared binding of a zinc atom seems to compensate adequately (3).The zinc ligand is tetrahedrally coordinated between Cys-176 of the L1 loop, His-179 of the helix in the L2 loop, Cys-238, and Cys-242 of the L3 loop.
DNA binds to p53 via four binding sites. Each binding site consists of four copies of a pentameter sequence PuPuPuC(A/T), where Pu is a purine nucleotide. Because sequences other than than pentameter sequence can also participate in binding, the structure of p53's DNA binding domain has to be flexible. To accomodate flexility in the DNA sequence, the primary structure of p53 is therefore able to hydrogen bond with base pairs other than the standard sequence. Because p53 checks DNA for errors in G1 of cell division, its most important functional residues are those that bind directly to the DNA. Zinc also plays a critical role in DNA binding—in addition to holding the loops of p53’s tertiary structure together in the absence of a strong hydrogen bond network, when wild-type p53 is treated with metal chelating agents, wild-type function is essentially inactivated.
Because p53 inhibits the growth of tumors by arresting DNA-damaged cells in G1 of cell division, oncogenic mutations in p53 are those at or near where DNA binds to the protein (3). The two most commonly mutated residues, Arg-248 and Arg-273, directly connect to the complexed DNA. When these mutations are expressed in the cell, p53 is unable to adequately check for DNA damage before allowing the cell to proceed with cell division, leading to neoplastic growth. The nature of these mutations is more than a simple loss of function. 85.6% of mutations in p53 are missense mutations, meaning that the change of a single nucleotide changes what amino acids the gene will then code for. For p53 protein, which has specific primary structure to best bind the DNA, this disrupts the tightness of connection between p53’s DNA binding domain and DNA as well as p53’s ability to bind the DNA. p53 differs from other tumor suppressor genes in this respect—while p53 tends towards missense mutations, other potential oncogenes tend to have higher rates of chain termination codons, deletions, exon-skipping mutations, or frame shift mutations. These missense mutations can also combine with other oncogenes to promote neoplastic growth.
Further insights about the structure of p53 are elucidated when comparing it to a related protein, p73. p73 (PDB ID: 4A63) is a tumor suppressor protein within the same family as p53 (4). p73 was determined to be an appropriate comparison structure for p53 through use of the Dali server and PSI-BLAST searches (5, 6). The Dali server uses a sum of pairs method to look for proteins with a similar three dimensional structure. The sum of pairs method works by comparing intramolecular distances; similarity between these distances in two structures is then described by a Z score (7). If two structures were significantly similar in structure, they would have a Z score above 2. p73 and p53 have a Z score of 32, which indicates very high similarity. BLAST is an NIH computer program that searches a protein databases for amino acid sequences similar to that of the protein of interest and assigns a value (the E score) to the similarity between two proteins. A lower E score indicates greater similarity. The E score for p73 and p53 is 6e-73, indicating high similarity; this is to be expected because p73 and p53 are members of the same protein family. Though the structures are very similar, p73 has a residue insertion that induces repacking around one of the most frequently mutated residues in p53, Arg-175, making p73 slightly less prone to oncogenic mutation. The DNA contact residues in p53 are strictly conserved in p73. The most structural deviation in the p73 structure occurs in the core β-sandwich, but this has little effect on DNA binding. p73 has a divergent loop that allows for more protein-protein interaction than p53. This divergent loop allows p73 to have more overall hydrogen bonding than p53, making p73 more thermodynamically stable.
Though p53 has been clearly established as a tumor suppressor protein, p73 does not fill the same function in the cell—it is rarely inactivated in human cancers as p53 is, but it does act in concert with p53 in cell cycle regulation, and thus has some tumor suppressor properties. Though the reasons behind p73’s disparate function are not entirely understood, some cancer-causing viruses (such as HPV and SV40) that inactivate p53 cannot bind to p73, likely because its increased hydrogen network makes these shifts in conformation energetically unfavorable, resulting in p73’s lower rate of inactivation in human cancers (8). However, because p73 plays a role in apoptosis of cells with damaged DNA, it is a useful drug target when p53 function is inactivated.
Due to p53’s role in tumor suppression, it also plays a role in cancer pharmacology. The p53 apoptosis pathway is a common target of cytotoxic agents intended to selectively kill tumor cells, though the exact mechanism of initiating this pathway via chemotherapy is not fully understood (9). When treating tumor cells with radiation or chemotherapeutic agents, the p53 pathway is essential in order to activate apoptosis. When the p53 pathway is absent or not expressed, tumors are at a much higher risk of being resistant to treatment. When treating tumor cells with chemotherapeutic agents, most colonies resistant to treatment are deficient in p53 function. In cells transformed by the oncogene E1A anticancer agents trigger the p53 apoptosis pathway and resistance to drugs is not observed. However, cells transformed by E1A but lacking p53 were resistant to the same cancer treating agents. Thus, though radiation and chemotherapy damages DNA, it is actually through the p53 apoptosis pathway that some chemotherapies result in tumor-specific cytotoxicity (10).
Cancer remains one of biomedical science’s biggest enigmas—even as scientists develop pharmacological methods of treating cancer, the pathways by which cancer arises and is cured are still somewhat of a mystery. The tumor suppressor protein p53, as both a clue into how cancer arises in humans and a snapshot into how it is eradicated, provides an invaluable avenue for further research. Though much is known about how p53 regulates cell growth and therefore also regulates tumor growth as well, its exact function and methods of initiating apoptosis are not fully understood. Further research on p53 and associated oncogenes could reveal mechanisms of cancerous transformation that lead to pathways for more effective cancer treatments (10).