Reversal of the DNA-Binding-Induced Loop L1 Conformational Switch in an Engineered Human p53 Protein

Abstract

The gene encoding the p53 tumor suppressor protein, a sequence-specific DNA binding transcription factor, is the most frequently mutated gene in human cancer. Crystal structures of homo-oligomerizing p53 polypeptides with specific DNA suggest that DNA binding is associated with a conformational switch. Specifically, in the absence of DNA, loop L1 of the p53 DNA binding domain adopts an extended conformation, whereas two p53 subunits switch to a recessed loop L1 conformation when bound to DNA as a tetramer. We previously designed a p53 protein, p53FG, with amino substitutions S121F and V122G targeting loop L1. These two substitutions enhanced the affinity of p53 for specific DNA yet, counterintuitively, decreased the residency time of p53 on DNA. Here, we confirmed these DNA binding properties of p53FG using a different method. We also determined by crystallography the structure of p53FG in its free state and bound to DNA as a tetramer. In the free state, loop L1 adopted a recessed conformation, whereas upon DNA binding, two subunits switched to the extended loop L1 conformation, resulting in a final structure that was very similar to that of wild-type p53 bound to DNA. Thus, altering the apo structure of p53 changed its DNA binding properties, even though the DNA-bound structure was not altered.

p53 Dynamically Directs TFIID Assembly on Target Gene Promoters

“…It is of note that photobleaching of the fluorescently labeled p53 did not affect our results, since similar residence times were observed with multiple dyes with differing levels of photostability (data not shown). In addition, a previous study detected similar short residence times of approximately 10 to 12 s for p53 bound to a consensus site (40). Collectively, these findings indicate that p53 dynamically interacts with DNA via both specific and nonspecific interactions.…”

Structural Basis for p53 Lys120-Acetylation-Dependent DNA-Binding Mode

“…It was also proposed that mutations that facilitate this conformational switch in loop L1 (e.g., S121F and V122G), also increase DNA binding specificity by increasing DNA-binding off rate and reducing the half-life of p53-DNA complexes [15,16]. Our data suggest that Lys120-acetylation facilitates the conformational switch in loop L1 upon binding to the consensus RE and not the BAX RE.…”

“…Our data suggest that Lys120-acetylation facilitates the conformational switch in loop L1 upon binding to the consensus RE and not the BAX RE. Following the model suggested by Halazonetis and coworkers [15,16], this difference in DNA binding modes may promote specific DNA binding by reducing the amount of time Lys120-acetylated p53 spends in interaction with random DNA or off-target REs, but not with the BAX RE.…”

“…tetramerization domain (p53-DBD+TET) in complex with DNA, recently published by Halazonetis and co-workers [14,15]. In the latter case, loop L1 of the outer subunits (B, C) was found in a significant recessed conformation, while loop L1 of the inner subunits (A, D) was found at the more common conformation in which Lys120 forms polar interactions with the DNA.…”

“…The central DBD is a 200 amino acid immunoglobuline-like β-sandwich structure responsible for sequence-specific DNA binding. Several structures of wild-type p53-DBD and its rescued mutants in a complex with DNA [6][7][8][9][10][11][12][13], as well as engineered mutants of p53 composed of the DBD and TET domains (p53-DBD+TET) in a complex with DNA [14][15][16], have been determined. According to these structures, p53-DBD binds specific DNA REs as a dimer of dimers, where each dimer recognizes half RE.…”