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p53 Core Domain Bound to DNA

Jane Robertson '08 and Allison Mauk '08


I. Introduction

As a transcription regulator, p53 plays a fundamental role in controling cell growth.  The activation of p53 consistantly results in apoptosis.  This function is necessary to help maintain healthy levels of cell proliferation.  When p53 becomes mutated, the ability to induce apoptosis is compromised.  Thus, p53 mutations frequently result in uncontrolled cell growth leading to tumors.  Therefore, understanding the structure, DNA-binding mechanism and mutational characteristics is of utmost importance within the medical community.

Overall, p53 is a large tetramer protein, with identical subunits.  Each subunit contains an N-terminal transactivation domain, a DNA-binding core domain (p53DBD), a tetramerization domain and a C-terminal regulatory domain [2].   However, due to the complexity of this protein, it has not been crystallized in its entirety.  This tutorial will focus on the DNA-binding core domain (p53DBD).  

The crystal structure obtained by Ho et al. was prepared from Mus musculus.  There are high levels of conserved sequence alignment bewteen the p53DBD of Mus musculus and the p53DBD that has been isolated in humans. Alignment The structural implications of the high level of sequence conservation between mice and humans can be observed when the two structures are overlayed. Overlay

II. General Structure

When the p53DBD is isolated from the rest of the p53 protein, it is found as a dimer . However, in the presence of the entire p53 complex and bound to DNA, it is suggested that the p53DBD undergoes a quaternary structure alteration to a dimer of dimers. Each of the subunits binds to the DNA nearly symmetrically and opposite of each other via two recognition helices . The dimerization contacts are formed through a Zinc binding domain which is positioned over the minor groove of the DNA At the site of dimerization, a 20° bend in the DNA is formed. However, this effect was not observed in a monomer of p53DBD that was crystallized bound to DNA. This suggests that it is the actual formation of the dimer that induces this bend

Each subunit is made up of a sandwich of antiparallel β-sheets and two α-helices . H1 is involved in the dimer-dimer interactions whereas H2 is responsible for DNA recognition and binding. There are also a number of loop regions, most importantly L2 and L3 . Another loop, the L1 region, shows a large amount of flexibility between species which has led to the suggestion that it is in no way involved with DNA binding. L1 In this particular paper, the L1 loop was unable to be crystallized which is likely a result of its flexibility. 

III. DNA Interactions

The contacts between p53DBD and DNA are mediated through the H2 recognition helix, the proceeding loop region, and the L3 loop. The alpha helix interacts with the DNA major groove at the decamer consensus sequence for the p53 recognition site.  The decamer consensus sequence for the recognition of the p53 dimer follows a very simple pattern of PuPuPuC(A/T|A/T)GPyPyPy, where Pu indicaties a purine and Py indicates a pyrimadine. All p53 response elements contain similar decamer recgonition sites.

Within the H2 helix, Arg 277 binds with guanosine 7 in the DNA major groove. In addition, Arg 277 makes DNA backbone contacts, as do Ala 276 and Arg 280. . Within the L3 loop region, Ser238 and Arg 245 interact with the DNA minor groove

IV. Dimer Interactions

As explained above, a zinc atom is fundamental in maintaining the structure of the p53 protein structure.  This zinc atom binds mainly to the H1 helix and L3 loop of the protein.  These two motifs are also fundamental in the dimer interactions that maintain the stability of the p53 dimer.  Dimer interactions involve van der Waals, hydrogen bonding, and electrostatic interactions. Van der Waals interactions are made between Pro-174 and His-174 from the H1 helices of opposing subunits of the dimer .  Additionally, there are intermolecular salt bridges resolved between Arg-178 and Glu-177 , both of which are also on the H1 helices of the dimer. there are hydrogen bonds between water molecules and Met-240.  The other bonds display dipole-dipole and electrostatic interactions between Arg-178 and Glu-177 on each subunit .

However, it is known the the p53 protein interacts with DNA as a dimer of dimers, or a tetramer.  Unfortunately, this structure has not yet been crystallized.  Based on the crystalized structure of the p53 dimer, Ho et al. created a model of the tetramer-DNA complex (Ho et al. Model). These two dimers interact in a tail to tail mechanism, whereas the DNA-bound dimer functions through head to head interactions.

IV. Mutations

p53 mutations in cancer cells can occur through various methods, including lesions that prevent p53 activation, mutations within TP53 gene, or mutations of downstream mediators of p53 function.  Regardless of how p53 becomes mutated, the mutation generally has drastic consequences. About half of all cancers display a p53 mutation[4].  

There are two classes of mutations that occur in p53: contact mutants and conformational mutants.  Affecting the amino acids required to maintain the structure of the p53 dimer, conformational mutants are found primarily in the H1 helix and L3 loop of the protein .  Contact mutants are mutations found near the protein-DNA interface, especially in the H2 helix .These are the residues most frequently found to be mutated in cancer. More specifically, Cho et al. determined there are six “hotspot” mutations . These residues—five arginines and one glycine—are the specific mutations most commonly seen in cancer. Arg-248 is the most common, accounting for 9.6% of p53 mutations . The other most commonly mutated residues are Arg-273 (8.8%) , Arg-175(6.1%) , Gly-245(6.0%) , Arg-249(5.6%) , and Arg-282(4.0%) [1]. Click here to reset: These six residues are all highly involved at the p53-DNA binding interface. Arg-248 makes contact at the minor groove, while Arg-273 contacts a phosphate on the backbone of the DNA. The four other residues are all involved in stablizing the structure of the DNA-binding surface of p53.

V. References

[1] Cho, Yunje, Svetlana Forina, Philip D. Jeffrey, Nikola P. Pavletich. 1994. Crystal Structure of a p53 Tumor Suppressor-DNA Complex: Understanding Tumorigenic Mutations. Science 265: 346-355.

[2] Ho, William C., Mary X. Fitxgerald, Ronen Marmorstein. 2006. Structure of the p53 Core Domain Dimer Bound to DNA. Journal of Biological Chemistry 281: 20494-20502.

[3] Nagaich, Akhelesh K., Victor B. Zhurkin, Steward R. Durell, Robert L. Jernigan, Ettore Apella, Rodney E. Harriington. 1999. p53-induced DNA bending and twisting: p53 tetramer binds on the outer side of a DNA loop and increases DNA twisting. Proc. Natl. Acad. Sci. 96: 1875-1880.

[4] Vousden, Karen H., Xin Lu. 2002. Live or Let Die: The Cell's Response to p53. Nature 2: 594-604.

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