Mutants of the tumour suppressor p53 L1 loop as second-site suppressors for restoring DNA binding to oncogenic p53 mutations: structural and biochemical insights

Abstract

To assess the potential of mutations from the L1 loop of the tumour suppressor p53 as second-site suppressors, the effect of H115N and S116M on the p53 'hot spot' mutations has been investigated using the double-mutant approach. The effects of these two mutants on the p53 hot spots in terms of thermal stability and DNA binding were evaluated. The results show that: (i) the p53 mutants H115N and S116M are thermally more stable than wild-type p53; (ii) H115N but not S116M is capable of rescuing the DNA binding of one of the most frequent p53 mutants in cancer, R248Q, as shown by binding of R248Q/H115N to gadd45 (the promoter of a gene involved in cell-cycle arrest); (iii) the double mutant R248Q/H115N is more stable than wild-type p53; (iv) the effect of H115N as a second-site suppressor to restore DNA-binding activity is specific to R248Q, but not to R248W; (v) molecular-dynamics simulations indicate that R248Q/H115N has a conformation similar to wild-type p53, which is distinct from that of R248Q. These findings could be exploited in designing strategies for cancer therapy to identify molecules that could mimic the effect of H115N in restoring function to oncogenic p53 mutants.

Structure and Stability Insights into Tumour Suppressor p53 Evolutionary Related Proteins

“…All the other structures stabilised at an rmsd of about 3.0 Å. A closer look at the local structure deviations responsible of these values can be found in the analysis of the loop 1 (L1) and loop 3 (L3) (Figure 5B and 5C), both of which are involved in DNA binding [62]. For all the studied proteins L1 seems to be more flexible and shows different motilities within the set.…”

Effects of p21-activated kinase 1 inhibition on 11q13-amplified ovarian cancer cells

“…Remarkably, we found that knockdown of Pak1, but not Pak2, led to G 2 /M cell cycle arrest, accompanied by activation of p53, strongly increased expression of p21 cip , and reduced expression of cyclin B1, consistent with the observed cell cycle effects. These findings were unexpected, as OVCAR-3 cells bear a R248Q mutation in p53 that reduces its ability to bind DNA and activate transcription (34, 35). We speculate that loss of Pak1 leads to phosphorylation and reactivation of this mutant p53 protein, partly restoring its function through a conformational change.…”

Molecular dynamics study of the conformational stability of esterase 2 from Alicyclobacillus acidocaldarius

“…MD Table 1 Percentage content of secondary structure elements in the two proteins at the end of the 20 ns MD trajectories, at the three investigated temperatures, determined by means of the STRIDE program [27]. simulations are a powerful approach to study biological processes that are not easily determinable by experimental techniques, and can provide useful insights into the molecular determinants driving the process [16,17]. The obtained results unequivocally indicate the presence, in the structure of EST2, of more flexible structural elements, the external ␣-helices, and of more resistant-rigid structural elements, the central twisted ␤-sheet, that are coupled by the last and long ␣-helix.…”