p53 protein-DNA complex

David Gold & Dawn Sokolowski '04

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I. Introduction

Cancer is caused by multiple DNA mutations that allow cells to proliferate without limit: both gain-of-function mutations that change proto-oncogenes into their active form, oncogenes, and loss of function mutations in tumor suppressor genes increase the likelihood for cancer (Alberts et al, 2003). p53  is a tumor suppressor gene that is mutated in about half of all cancers (Alberts et al, 2003). p53 is not necessary for development because p53 knock-out mice develop normally; however, the knock-out mice are prone to cancer and develop it within three months (Donehower et al, 1992). Under stress conditions, p53 expression increases to induce apoptosis or to arrest the cell cycle allowing ample time for DNA repair (Bálint et al, 2001). In addition, p53 is involved in multiple types of DNA damage repair such as nucleotide excision repair, base excision repair and correction of double strand breaks (Bálint et al, 2001).

When p53 function is reduced due to mutations that often occur prior to cancer formation, DNA repair capabilities are reduced: p53 can no longer aid in DNA restoration or arrest the cell cycle to provide time for other factors to correct DNA damage. Apoptosis would normally remove cells with damaged DNA; however, loss of p53 function also results in a decreased ability of the cell to undergo apoptosis. Without apoptosis, the cell continues to proliferate despite severe DNA damage, promoting cancer formation. Mutations in p53 along with mutations in other proto-oncogenes and tumor-suppressor genes are unfortunately very common and often result in cancer. Because of the multiple roles of p53, loss of p53 function in cells is deleterious and unfortunately very common in cancer (Alberts et al, 2003).

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II. General Structure

p53 is a tetramer consisting of four similar polypeptide chains. A trimer of the p53 complex (residues 94-312) was co-crystallized with a 21 bp DNA duplex containing half of the p53 binding site, which is sufficient for binding. It is thought that crystal-packing forces inhibit the fourth subunit from binding (Cho et al, 1994). The first subunit  binds a consensus site on the DNA duplex and forms contacts to the bases and backbone. The second subunit  binds 11 bp away in a region of modest homology, weakly interacting with the first subunit in a head-to-tail dimer. The third subunit  does not bind DNA. However, it makes protein-protein contacts that stabilize crystal packing. DNA binding does not induce structural changes to the protein.

The core domains each consist of two anti-parallel beta-sheets that contain four to five beta-strands  . The core domains also contain a loop-sheet-helix motif  packed across an extended hydrophobic core, forming a beta sandwich. Two large loops at the edge of the sandwich are bound together by a tetrehedrally coordinated zinc atom   . The helix and loop bind to the major groove and contact the edges of base pairs. Both structures contact the DNA backbone. The amino terminus of each subunit consists of five tightly packed beta strands. The carboxyl terminus consists of four beta strands connected by short 5-11 residue loops. .

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III. DNA binding domain

p53 binding sites contain four copies of the pentameric consensus sequence PuPuPuC(A/T). Residues 102-292 of p53 bind DNA with sequence specificity. Protein-DNA interactions occur at the major and minor groves and at the DNA backbone.

In the major groove  , Lys120 from loop L1 hydrogen bonds with G8 while Cys277 hydrogen bonds with C9. The most important major grove contact is between Arg280 from the a-helix to G10. In addition, Arg280 is stabilized by a salt bridge with the Asp281 carboxylate.

In the minor groove  , Arg248from loop L3 is packed against the DNA backbone because of the local compression of the minor groove. At this region, the minor groove width is 9.3Å rather than the normal 11.5Å of B-DNA. This probably results from the high A-T content of the sequence. A water molecule makes hydrogen bonds between Arg248 and G13.

DNA backbone contacts  occur between the phosphate of G10 and Ser241 from the L3 loop and Ala276 from the loop-sheet-helix. The phosphate of T11 is bound to Arg273. Arg273 is also involved in multiple interactions including a salt bridge with the carboxylate of Asp281. In addition, Lys120 contacts the O3’ of T6 while Arg283 contacts the phosphate of G7.

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IV. Common Mutations

The DNA binding domain is the most conserved region of p53, yet deleterious mutations in this domain are the most frequent (Walker et al, 1999). The loop-sheet-helix motif and the two large loops that bind DNA correspond to highly mutable regions of the core domain. Mutations within these regions are implicated in oncogene activation and formation of several forms of cancer (Almog and Rotter, 1998). These mutations inactivate p53 by eliminating important DNA binding contacts or altering the structural stability of the core domain. Mutations are most frequent within the L3 loop, the L2 loop, the H2 alpha-helix and the C-terminus portion of the S10 strand   .

The most frequently mutated residues are Arg-175, Arg-248, Arg-249, Arg-282, and Gly-245. Arg-175 bridges portions of the L2 and L3 loops, stabilizing them   . Arg-175 mutants unfold portions of the core domain, creating structural defects that allow accessibility of proteases to the N-terminus (Cho et al, 1994). Arg-273 and Arg-280 are mutations in the DNA binding domain and limit p53 DNA binding by disrupting phosphate backbone linkages  ..

V. References

Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. (2003) Molecular Biology of the Cell, 4th ed. Garland Science, New York, NY. 1463pp.

Almog N, Rotter V. (1998) An insight into the life of p53: a protein coping with many function! Biochimica et Biophisica Acta. 1378: R43-R54.

Bálint É, Vousden KH. (2001) Activation and activities of the p53 tumor suppressor protein. British Journal of Cancer 85(12): 1813-1823.

Cho Y, Gorina S, Jeffrey PD, Pavletich NP. (1994) Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations. Science 265: 346-355.

Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery CA Jr, Butel JS, Bradley A (1992) Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356:215-221

Walker DR, Bond JP, Tarone RE, Harris CC, Makalowski W, Boguski MS, Greenblatt MS. (1999) Evolutionary conservation and somatic mutation hotspot maps of p53: correlation with p53 protein structural and functional features. Oncogene 19:211-218.

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