Op-nare130631 8748..8759

A p53 hot-spot mutation found frequently in human cancer is the replacement of R273 by histidine or cysteine residues resulting in p53 loss of function as a tumor suppressor. These mutants can be reactivated by the incorporation of second-site suppressor mutations. Here, we present high-resolution crystal structures of the p53 core domains of the cancer-related proteins, the rescued proteins and their complexes with DNA. The structures show that inactivation of p53 results from the incapacity of the mutated residues to form stabilizing interactions with the DNA backbone, and that reactivation is achieved through alternative interactions formed by the suppressor mutations. Detailed structural and computational analysis demonstrates that the rescued p53 complexes are not fully restored in terms of DNA structure and its interface with p53. Contrary to our previously studied wild-type (wt) p53-DNA complexes showing non-canonical Hoogsteen A/T base pairs of the DNA helix that lead to local minor-groove narrowing and enhanced electrostatic interactions with p53, the current structures display Watson–Crick base pairs associated with direct or water-mediated hydrogen bonds with p53 at the minor groove. These findings highlight the pivotal role played by R273 residues in supporting the unique geometry of the DNA target and its sequence-specific complex with p53. INTRODUCTION The tumor suppressor p53 acts as a DNA sequencespecific transcription factor regulating and activating the expression of a range of target genes in response to genotoxic stress. This in turn initiates a cascade of signal transduction pathways leading to different cellular responses including cell-cycle arrest and apoptosis that are known to prevent cancer development (1–4). p53 binds as a tetramer to specific response elements consisting mainly of two decameric half-sites separated by a variable number of base pairs (5–7). Mutations in the p53 gene that lead to inactivation of the protein are observed in 50% of human cancers (8,9). The majority of tumor-related p53 mutations, particularly those defined as mutational ‘hotspots’, occur within the DNA-binding core domain of p53 (10). The top hotspot mutations are located at or near the protein–DNA interface and can be divided into two major groups: DNA-contact mutations affecting residues involved directly in DNA contacts without altering p53 conformation, and structural mutations that cause a conformational change in the core domain (11). R273, a DNA-contact amino acid, is one of the most frequently altered residues in human cancer (6.4% of all somatic mutations), with mutations to histidine (46.6%) and to cysteine (39.1%) being most common (8,9). Crystal structures of the p53 core-domain bound to DNA (12–17) show that the positively charged guanidinium groups of R273 residues interact with the negatively charged DNA backbone at the center of each DNA half-site, supported by salt-bridge and hydrogenbond interactions. As discussed previously, R273 residues play a pivotal role in docking p53 to the DNA *To whom correspondence should be addressed. Tel: +972 8 934 2672; Fax: +972 8 934 6278; Email: [email protected] The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. 8748–8759 Nucleic Acids Research, 2013, Vol. 41, No. 18 Published online 17 July 2013 doi:10.1093/nar/gkt630 The Author(s) 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. at U niersity of Sothern C alornia on O cber 5, 2013 http://narrdjournals.org/ D ow nladed from at U niersity of Sothern C alornia on O cber 5, 2013 http://narrdjournals.org/ D ow nladed from at U niersity of Sothern C alornia on O cber 5, 2013 http://narrdjournals.org/ D ow nladed from at U niersity of Sothern C alornia on O cber 5, 2013 http://narrdjournals.org/ D ow nladed from at U niersity of Sothern C alornia on O cber 5, 2013 http://narrdjournals.org/ D ow nladed from at U niersity of Sothern C alornia on O cber 5, 2013 http://narrdjournals.org/ D ow nladed from at U niersity of Sothern C alornia on O cber 5, 2013 http://narrdjournals.org/ D ow nladed from at U niersity of Sothern C alornia on O cber 5, 2013 http://narrdjournals.org/ D ow nladed from at U niersity of Sothern C alornia on O cber 5, 2013 http://narrdjournals.org/ D ow nladed from at U niersity of Sothern C alornia on O cber 5, 2013 http://narrdjournals.org/ D ow nladed from at U niersity of Sothern C alornia on O cber 5, 2013 http://narrdjournals.org/ D ow nladed from at U niersity of Sothern C alornia on O cber 5, 2013 http://narrdjournals.org/ D ow nladed from backbone at the central region of each half-site where no direct base-mediated contacts exist (13). Substitution of R273 by histidine or cysteine, referred to as R273H and R273C, leads to dramatic reduction in the DNA binding affinity, even though the protein retains wild-type stability (18). The reactivation of mutant p53 by various pathways faces a common challenge: reversing the effect of a single amino acid mutation in the core domain, thus restoring its natural function. It has been shown by DNA binding, transcriptional activation and tumor-suppressing assays that the incorporation of a second mutation into oncogenic p53, referred to as second-site suppressor mutation, can rescue the normal activity of p53 as described later in the text for the current hot-spot mutations. In the case of R273H and R273C, it was shown that the replacement of threonine by arginine at position 284 (T284R) restores activity to both p53 mutants (19). Replacing serine by arginine at position 240 (S240R) was also found to rescue R273H (20). Although S240R alone was found to act as a suppressor mutation only for R273H, it was shown that in combination with either T123A or H178Y, S240R can suppress the effect of R273C mutation on p53 function (20). These observations and the fact that both R273H and R273C mutations have similar effects on p53 structure and function (21) suggest that S240R alone might also rescue R273C. In a more recent screen of p53 second-site suppressor mutations, R273H was also shown to be rescued by H178Y (22). To elucidate the structural basis of p53 dysfunction as a result of DNA-contact mutations and the mechanisms of their rescue by second-site suppressor mutations, we crystallized and analyzed the structures of the corresponding single and double p53 mutants using the core domain of wild-type human p53 as a template. These include the oncogenic mutants R273H and R273C, the rescued proteins harboring each of the aforementioned mutations together with a second-site suppressor mutation, T284R or S240R, and their sequence-specific complexes with consensus DNAbinding sites. The comparative analysis of the various structures shows that inactivation of the cancer-related mutants results from the lack of stabilizing interactions between p53 and the DNA target. Reactivation of these proteins is achieved by new interactions formed by the second-site mutations, T284R or S240R, with the DNA backbone. However, the protein–DNA interface at the center of each DNA half-site is distinctly different from that of the wt complex in terms of DNA shape and minorgroove interactions. MATERIALS AND METHODS