Hot-spot mutants of p53 core domain evince characteristic local structural changes

TL;DR

NMR spectroscopy of the p53 core domain shows that five common hot-spot mutants cause characteristic local structural changes rather than global unfolding. R248Q, often considered only a DNA-contact mutant, also perturbs the L2–L3 region, while V143A produces long-range changes in the β-sandwich and DNA-binding surface.

Key findings

• Of 200 backbone amide signals, 176 were assigned; solution secondary structure matched the crystal structure, and residues 294–312 had random-coil shifts, indicating an unstructured C-terminal tail.
• G245S, R248Q, R249S, and R273H showed only localized chemical-shift changes, suggesting overall folds similar to wild type. R273H changes mapped mainly to the loop–sheet–helix motif and L3, including residues 281, 282, 285, and 286 after loss of the Arg-273–Asp-281 salt bridge.
• L3 mutations G245S, R248Q, and R249S introduced changes in L2 and L3 and terminal residues of strands 4, 9, and 10. R248Q produced changes as extensive as R249S; both are ≈2 kcal/mol less stable than wild type.
• V143A caused chemical-shift changes in almost all residues of the β-sandwich and the DNA-binding surface, which the authors suggest may affect DNA-binding specificity.

Methods

• Solution NMR compared wild-type and five mutant human p53 core domains (residues 94–312): V143A, G245S, R248Q, R249S, and R273H.
• Samples included 15N-labeled proteins and an 80% 2H, uniformly 13C,15N-labeled sample; 1H–15N HSQC spectra were acquired at 293 K, and triple-resonance experiments at 298 K on Bruker 600- and 800-MHz spectrometers.
• Backbone assignments used triple-resonance experiments and NOEs; structural changes were monitored by chemical-shift changes, with secondary structure assessed from ΔCα − ΔCβ deviations from random-coil values.

Limitations

• Stated in the paper: whether PAb240 can bind a partly unfolded form is still under active debate, and the authors note that comparing backbone dynamics of wild type and hot-spot mutants “may prove informative.”
• Evident from the text: conclusions rest on chemical-shift changes rather than full high-resolution structures of the mutants.
• Evident from the text: only five hot-spot mutants were studied, and the NMR measurements were performed at 20°C/293 K without direct DNA-binding or functional assays in this paper.