Mapping Interactions of the Intrinsically Disordered C-Terminal Regions of Tetrameric p53 by Segmental Isotope Labeling and NMR

TL;DR

Using trans-intein splicing and segmental ¹⁵N labeling, the authors obtained atomic-level NMR data on the disordered C-terminal regions of full-length tetrameric p53. They show that the CTD dynamically binds DNA flanking the p53 recognition element, and that acetylation of K372/K373/K382 impairs this interaction.

Key findings

• The NLS and CTD remain largely disordered and highly dynamic in full-length p53; TET-domain resonances (Y327–G356) are broadened beyond detection in the full-length tetramer but are visible in the isolated p53(304–393) peptide.
• The CTD contacts DNA outside the 20-bp p53 recognition element: residues 369–374 and 381–388 show substantial chemical shift changes with a 60-bp DNA (DNA-L) but only minor changes with the 20-bp DNA-S. These interactions increase as flanking DNA length increases and level off beyond ~20 bp (~70 Å) on either side of the recognition element.
• CTD–DNA binding is sequence independent: similar CTD chemical shift changes occur with ATG-repeat flanks, random flanks, and native p21 flanks.
• Acetylation by CBP(1079–1855) at K372, K373, and K382 (confirmed by Western blot) impairs but does not completely abrogate CTD binding to DNA; K305 also showed chemical shift changes suggesting acetylation.

Methods

• Full-length p53 constructs contained the quadruple M133L/V203A/N239Y/N268D superstabilizing mutations; segmental labeling used Npu DnaE trans-intein splicing to produce p53 labeled with ¹⁵N at residues 304–393 [¹⁵N(304–393)-p53].
• NMR experiments included ¹H-¹⁵N HSQC, TROSY-HSQC, and SOFAST-HMQC at 25°C in 20 mM Tris, pH 7.0, 150 mM NaCl, 2 mM DTT, 5% D₂O; protein concentrations ranged from 30–150 μM. Backbone assignments for p53(304–393) were deposited in the BMRB under accession 51613.
• DNA constructs ranged from 20–80 bp, with a central 20-bp p53 recognition element flanked by 0–30 bp of (ATG)n or other sequences; p53:DNA complexes used a 4:1 protomer:DNA ratio (1:1 tetramer:DNA). Acetylation used CBP(1079–1855) at a 100:1 p53:CBP molar ratio with 1 mM acetyl-CoA.

Limitations

• The paper states that TET-domain backbone amide resonances are broadened beyond detection in full-length p53, so the authors cannot comment on potential TET:DNA interactions.
• The paper notes that four non-native residues (CFNG) are inserted at the splice site between S303/T304, causing substantial chemical shift differences for residues adjacent to the splice site.
• Evident from the text: all structural and interaction data are from in vitro purified-protein NMR experiments under defined buffer conditions, so the cellular context and physiological consequences of the observed CTD–DNA and acetylation effects are not directly measured.