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Updated: Jan 29, 2026
Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO
Published on: December 28, 2019
The C-terminal SAM domain of p73 binds to the N terminus of MDM2
José L Neira1, Clara Díaz-García2, Manuel Prieto2
1Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche, Alicante, Spain; Instituto de Biocomputación y Física de Sistemas Complejos, Joint Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Universidad de Zaragoza, 50009 Zaragoza, Spain.
Background:
The p53, p63 and p73 proteins belong to the p53 family of transcription factors, playing key roles in tumour suppression. The α-splice variant of p73 (p73α) has at its C terminus a sterile alpha motif (SAM); this domain, SAMp73, formed by five helices (α1 to α5), is thought to mediate in protein-protein interactions. The E3-ligase MDM2 binds to p73 at its N terminus transactivation domain (TA), but it does not promote its degradation via ubiquitination; however, the details of such MDM2/p73 interaction are not fully known.
Methods:
We studied the binding of SAMp73 with N-terminal MDM2, by several biophysical techniques, namely, fluorescence, far-UV circular dichroism (CD), NMR and bio-layer interferometry (BLI).
Results:
Our results obtained by fluorescence, T2-relaxation measurements and BLI show that there was binding between both proteins with a dissociation constant of ~10 μM. Furthermore, the binding region of SAMp73 involved mainly residues in the major α-helix, α5, and the nearby α4, as shown by HSQC-NMR. The binding was so specific that an isolated peptide comprising α4 and α5 helices of SAMp73, α4α5, did also bind to the N terminus of MDM2, although with weaker affinity than the entire domain.
Conclusions:
A new interaction between MDM2 and SAMp73 has been found, which could have potential therapeutic applications in cancers involving inactivated p53.
General Significance:
A novel interaction between the C-terminal SAM of p73 and N-terminal MDM2 is described. The interaction could be used to modulate the functions where the wild-type, intact p73 is involved.
Insights
Researchers discovered a new interaction between MDM2 and the sterile alpha motif (SAM) domain of p73 (SAMp73). This finding could lead to new cancer therapies targeting p53 family proteins.
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- The p53 family, including p53, p63, and p73, are crucial tumor suppressors.
- The p73 alpha splice variant (p73α) contains a sterile alpha motif (SAM) domain (SAMp73) involved in protein interactions.
- MDM2, an E3 ligase, interacts with p73 but does not typically cause its degradation, with details of this interaction remaining unclear.
Purpose of the Study:
- To investigate the binding interaction between SAMp73 and the N-terminal domain of MDM2.
- To characterize the biophysical properties and binding interface of the MDM2-SAMp73 complex.
Main Methods:
- Fluorescence spectroscopy
- Far-UV circular dichroism (CD)
- Nuclear Magnetic Resonance (NMR) spectroscopy (specifically HSQC-NMR and T2-relaxation measurements)
- Bio-layer interferometry (BLI)
Main Results:
- Biophysical techniques confirmed binding between MDM2 and SAMp73 with a dissociation constant of approximately 10 μM.
- NMR studies identified the binding interface on SAMp73, primarily involving residues in the α5 and α4 helices.
- A specific peptide (α4α5) derived from SAMp73 also bound to N-terminal MDM2, albeit with lower affinity.
Conclusions:
- A novel interaction between the C-terminal SAM domain of p73 and the N-terminal domain of MDM2 has been elucidated.
- This newly identified interaction offers potential therapeutic strategies for cancers associated with inactivated p53, by modulating p73 functions.
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