Structural Basis of Mutation-Dependent p53 Tetramerization Deficiency

Abstract

The formation of a tetrameric assembly is essential for the ability of the tumor suppressor protein p53 to act as a transcription factor. Such a quaternary conformation is driven by a specific tetramerization domain, separated from the central DNA-binding domain by a flexible linker. Despite the distance, functional crosstalk between the two domains has been reported. This phenomenon can explain the pathogenicity of some inherited or somatically acquired mutations in the tetramerization domain, including the widespread R337H missense mutation present in the population in south Brazil. In this work, we combined computational predictions through extended all-atom molecular dynamics simulations with functional assays in a genetically defined yeast-based model system to reveal structural features of p53 tetramerization domains and their transactivation capacity and specificity. In addition to the germline and cancer-associated R337H and R337C, other rationally designed missense mutations targeting a significant salt-bridge interaction that stabilizes the p53 tetramerization domain were studied (i.e., R337D, D352R, and the double-mutation R337D plus D352R). The simulations revealed a destabilizing effect of the pathogenic mutations within the p53 tetramerization domain and highlighted the importance of electrostatic interactions between residues 337 and 352. The transactivation assay, performed in yeast by tuning the expression of wild-type and mutant p53 proteins, revealed that p53 tetramerization mutations could decrease the transactivation potential and alter transactivation specificity, in particular by better tolerating negative features in weak DNA-binding sites. These results establish the effect of naturally occurring variations at positions 337 and 352 on p53’s conformational stability and function.

How Do Cancer-Related Mutations Affect the Oligomerisation State of the p53 Tetramerisation Domain?

“…0.9 ppm. The 1 H, 15 N HSQC spectrum of 37TD-L344P, obtained at natural abundance, showed a similar lack of signal dispersion for the amide NH resonances (Supplementary Materials, Figure S33), thereby further confirming the unstructured nature of this peptide. In the 1 H, 13 C-HSQC spectra of 37TD-L344P (Figure 5A), a single cross-peak resonating at 2.05 ppm was detected, close to the expected value (2.10 ppm) for a random coil peptide [68].…”

“…R337H is the most common mutation in the TD, and it is associated with Li-Fraumeni syndrome, which gives rise to several types of cancer. Due to its correlation with cancer, this mutant has been studied and characterised by several groups [ 55 , 56 , 57 , 58 , 59 ], as well as in yeast [ 15 ] and mouse [ 60 ] models. Di Giammarino and co-workers reported that R337H strongly destabilises the tetramer and that its stability is pH-dependent, i.e., tetramer stability is partially recovered by decreasing the pH to acidic values [ 55 ].…”

“…The oligomerisation of p53 may occur stepwise in different times and areas of the cell. In particular, dimerisation occurs co-translationally on the polysome, whereas tetramerisation is a posttranslational event that takes place in the nucleus [ 14 , 15 ]. Appropriate subcellular localisation is modulated by specific sequences within the C -terminal region [ 16 , 17 ].…”