Acetylation of lysine 120 of p53 endows DNA-binding specificity at effective physiological salt concentration

Abstract

Lys120 in the DNA-binding domain (DBD) of p53 becomes acetylated in response to DNA damage. But, the role and effects of acetylation are obscure. We prepared p53 specifically acetylated at Lys120, AcK120p53, by in vivo incorporation of acetylated lysine to study biophysical and structural consequences of acetylation that may shed light on its biological role. Acetylation had no affect on the overall crystal structure of the DBD at 1.9-Å resolution, but significantly altered the effects of salt concentration on specificity of DNA binding. p53 binds DNA randomly in vitro at effective physiological salt concentration and does not bind specifically to DNA or distinguish among its different response elements until higher salt concentrations. But, on acetylation, AcK120p53 exhibited specific DNA binding and discriminated among response elements at effective physiological salt concentration. AcK120p53 and p53 had the highest affinity to the same DNA sequence, although acetylation reduced the importance of the consensus C and G at positions 4 and 7, respectively. Mass spectrometry of p53 and AcK120p53 DBDs bound to DNA showed they preferentially segregated into complexes that were either DNAðp53DBDÞ 4 or DNA ðAcK120DBDÞ 4 , indicating that the different DBDs prefer different quaternary structures. These results are consistent with electron microscopy observations that p53 binds to nonspecific DNA in different, relaxed, quaternary states from those bound to specific sequences. Evidence is accumulating that p53 can be sequestered by random DNA, and target search requires acetylation of Lys120 and/or interaction with other factors to impose specificity of binding via modulating changes in quaternary structure.

Nuclear interactor of ARF and Mdm2 regulates multiple pathways to activate p53

“…28,29 Acetylation of K120 alters the DNA binding specificity of p53, favoring its accumulation on low-affinity apoptotic gene promoters, such as bax and puma, triggering cell death. 28,29,46,47 We found that wild-type NIAM can promote p53 acetylation at K120. However, the N-terminal (NT) form lacks the ability to promote p53 acetylation despite retaining wild-type or greater abilities to bind Tip60, inhibit proliferation, and induce p53 transactivation of the p21 promoter.…”

Differential effects on p53-mediated cell cycle arrest vs. apoptosis by p90

“…Acetylation at K120 is indispensable for activation of proapoptotic targets but is not required for activation of growth-arrest targets (16,17). Although the mechanism underlying this target specificity remains to be elucidated, it is possible that acetylation at K120 may impose specificity through altering the p53 quaternary structure and thus endowing p53 binding to low-affinity response elements that are found on proapoptotic promoters (29,30). We also noticed a small amount of cytoplasmic p53-p90 interaction.…”

Crystal Structures of the DNA-binding Domain Tetramer of the p53 Tumor Suppressor Family Member p73 Bound to Different Full-site Response Elements

“…In the case of p53, acetylation of Lys-120 by acetyltransferases of the MYST family has been shown to be important to distinguish between the apoptotic cell response and cell arrest (40,41). Moreover, biochemically, it has been demonstrated that Lys-120 is important for DNA binding and that its acetylation increases the DNA specificity of p53 (42)(43)(44). In the case of p73, Lys-138 (where we observed different conformations depending on the RE sequence) is the conserved equivalent residue of Lys-120 in p53.…”