Abstract
Conformationally flexible protein complexes represent a major challenge for structural and dynamical studies. We present herein a method based on a hybrid NMR/MD approach to characterize the complex formed between the disordered p53TAD1–60 and the metastasis‐associated S100A4. Disorder‐to‐order transitions of both TAD1 and TAD2 subdomains upon interaction is detected. Still, p53TAD1–60 remains highly flexible in the bound form, with residues L26, M40, and W53 being anchored to identical hydrophobic pockets of the S100A4 monomer chains. In the resulting “fuzzy” complex, the clamp‐like binding of p53TAD1–60 relies on specific hydrophobic anchors and on the existence of extended flexible segments. Our results demonstrate that structural and dynamical NMR parameters (cumulative Δδ, SSP, temperature coefficients, relaxation time, hetNOE) combined with MD simulations can be used to build a structural model even if, due to high flexibility, the classical solution structure calculation is not possible.
Highlights
Structural information is obtained from chemical shift values, resonance assignment based on triple resonance measurements, for both free and bound p53TAD1–60
The complex formation between 15Nlabeled p53TAD1–60 and unlabeled Ca2 + -S100A4 is monitored by chemical-shift mapping from 1H,15N HSQC spectra
P53TAD1–60 residues involved in complex formation are revealed by calculating the variation of individual chemical shifts upon complex formation represented by the cumulative Δδ values.[41]
Summary
A physical and functional interaction between the metastasis related S100A4 protein and p53 was suggested for the first time by Grigorian et al.[32] and was further investigated in vitro.[33]. A strong inverse correlation between S100A4 and p53 has been shown by immunohistochemistry in lung adenocarcinoma, suggesting that the level of S100A4 is higher in wild-type p53 tumors.[36] A trend toward inverse correlation between S100A4 and p53 was shown in a breast cancer cohort, where a higher level of S100A4 was found to be a negative prognostic factor.[37] the importance of this complex lies in its potential role in the survival of cancer cells.[32,38,39] Despite all these, only limited structural information is available. The latest effort by X-ray crystallography was successful only if a shorter p53TAD17–56 segment was covalently bound to the N terminus of a C-terminally truncated S100A4Δ8 in the presence of a crystallization chaperon protein covalently linked to the complex.[26] In the present work we modeled the solution structure of this complex using full length p53TAD1–60 and wild-type S100A4. S100A4-p53 interaction aim to clarify the function and the effects of this association
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