Long-range regulation of p53 DNA binding by its intrinsically disordered N-terminal transactivation domain

Abstract

Atomic resolution characterization of the full-length p53 tetramer has been hampered by its size and the presence of extensive intrinsically disordered regions at both the N and C termini. As a consequence, the structural characteristics and dynamics of the disordered regions are poorly understood within the context of the intact p53 tetramer. Here we apply trans-intein splicing to generate segmentally 15N-labeled full-length p53 constructs in which only the resonances of the N-terminal transactivation domain (NTAD) are visible in NMR spectra, allowing us to observe this region of p53 with unprecedented detail within the tetramer. The N-terminal region is dynamically disordered in the full-length p53 tetramer, fluctuating between states in which it is free and fully exposed to solvent and states in which it makes transient contacts with the DNA-binding domain (DBD). Chemical-shift changes and paramagnetic spin-labeling experiments reveal that the amphipathic AD1 and AD2 motifs of the NTAD interact with the DNA-binding surface of the DBD through primarily electrostatic interactions. Importantly, this interaction inhibits binding of nonspecific DNA to the DBD while having no effect on binding to a specific p53 recognition element. We conclude that the NTAD:DBD interaction functions to enhance selectivity toward target genes by inhibiting binding to nonspecific sites in genomic DNA. This work provides some of the highest-resolution data on the disordered N terminus of the nearly 180-kDa full-length p53 tetramer and demonstrates a regulatory mechanism by which the N terminus of p53 transiently interacts with the DBD to enhance target site discrimination.

Identifying molecular features that are associated with biological function of intrinsically disordered protein regions

“…Because Mfg1 contains three predicted IDRs (and in general proteins contain multiple IDRs), this illustrates the advantage of our approach that infers which (if any) of the IDRs are associated with a particular function. Although in some well-studied cases, it is clear that the C-terminal and N-terminal IDRs have different functions (e.g., p53, (Krois et al, 2018;Laptenko et al, 2016)), in general we do not know if functional information within IDRs is modular (confined to one IDR) or distributed (spread over all the IDRs in a protein). In our exploration of yeast IDRs strongly associated with functions, we identified interesting cases where multiple IDRs within a protein were strongly associated with different functions (e.g., Supplementary figure S3) possibly supporting the idea that IDRs can be thought of as functional modules.…”

Single molecule studies reveal that p53 tetramers dynamically bind response elements containing one or two half sites

“…There are no high resolution structures of full length p53 bound to DNA, likely due to the unstructured N-and C-terminal regions interfering with structure determination. Because these domains impact the ability of p53 to bind DNA (15,19,20,36), it reasonable to predict these regions could impact the structure of p53/DNA complexes.…”

“…Our understanding of how p53 binds its RE has been informed from numerous important biochemical and structural studies that describe unifying principles (14); however inconsistencies and questions remain. A contributing factor is the need for more structural and quantitative biochemical studies using the full length p53 protein, particularly in light of evidence showing that regions outside the DNA binding domain can impact the interaction of p53 with DNA (15)(16)(17)(18)(19)(20).…”

“…The N-terminal region contains two unstructured transcriptional activation domains that interact with co-regulatory complexes to mediate transcriptional activation (21). Recent work revealed that intramolecular interactions between the N-terminal region and the DBD can impact the affinity and specificity with which p53 binds DNA (19,20). The DBD is largely responsible for recognizing and making contacts with p53 REs.…”

Common Functions of Disordered Proteins across Evolutionary Distant Organisms

“…Much like the TAD of human tumour suppressor p53, the VP16 TAD contains two disordered activation subdomains, each with transcription activation potential that may form amphipathic α-helices upon complex formation [191,192]. Recently, Krois and co-workers gave remarkable structural insights into the binding specificity of p53 by showing how the p53 TADs directly compete with non-specific DNA sequences for binding to the DNA-binding core domain [193]. As neither VP16 nor p53 are present in the genome of vascular plants, the VP16 design principles were used to screen for TADs of plant regulatory regions [194].…”