The Crystal Structure of the R280K Mutant of Human p53 Explains the Loss of DNA Binding

Abstract

The p53 tumor suppressor is widely found to be mutated in human cancer. This protein is regarded as a molecular hub regulating different cell responses, namely cell death. Compelling data have demonstrated that the impairment of p53 activity correlates with tumor development and maintenance. For these reasons, the reactivation of p53 function is regarded as a promising strategy to halt cancer. In the present work, the recombinant mutant p53R280K DNA binding domain (DBD) was produced for the first time, and its crystal structure was determined in the absence of DNA to a resolution of 2.0 Å. The solved structure contains four molecules in the asymmetric unit, four zinc(II) ions, and 336 water molecules. The structure was compared with the wild-type p53 DBD structure, isolated and in complex with DNA. These comparisons contributed to a deeper understanding of the mutant p53R280K structure, as well as the loss of DNA binding related to halted transcriptional activity. The structural information derived may also contribute to the rational design of mutant p53 reactivating molecules with potential application in cancer treatment.

SLMP53-1 interacts with wild-type and mutant p53 DNA-binding domain and reactivates multiple hotspot mutations

“…2A). Moreover, as reported [12] in mutp53 R280K, the arginine to lysine substitution abolishes these direct interactions with DNA ( Fig. 2A), and no substantial differences were found in the structural motifs' conformational rearrangements.…”

“…The PCR products and the mammalian expression vector pcDNA3 (Invitrogen, Alfagene, Lisboa, Portugal) were digested with KpnI/XhoI (New England Biolabs, Werfen), purified from agarose gel, and ligated with T4 DNA ligase (Promega, VWR, Carnaxide, Portugal), originating the expression vectors pcDNA3-mutp53. These constructs were propagated in NZY5α E. coli cells (NZYTech, Lisboa, Portugal) as described [12]. The sequence of each mutp53 in the constructed vectors was confirmed by sequencing (Eurofins GATC Biotech, Konstanz, Germany) with specific pcDNA3 primers.…”

The Conserved Arginine Required for AvrRps4 Processing Is Also Required for Recognition of Its N-Terminal Fragment in Lettuce

“…Similarly, the R280 to lysine mutation abolishes the DNA binding ability of human p53 protein, a well-known tumor suppressor (Malcikova et al, 2010). According to a structure study (Gomes et al, 2018), the interaction between R280 and the guanine nucleotide is stronger than that of lysine as it forms two hydrogen bonds, while lysine can only form one hydrogen bond. In addition, the side chain of arginine is longer and is more positively charged than lysine, which favors the interaction of p53 with the DNA molecule (Gomes et al, 2018).…”

Genome Instability and Pressure on Non-Homologous end Joining drives Chemotherapy Resistance via a DNA Repair Crisis Switch in Triple Negative Breast Cancer.

“…In our study, the lines that achieved the highest levels of resistance, MDA-MB-231 and BT549, have R280K and R249S p53 gain-of-function missense mutations, respectively. These mutations reduce DNA binding and infers reduced activation of cell death pathways (28,29). Cancer cells that harbor mutation of p53 modulate induction of apoptosis through p73 (30).…”