The C-terminal SAM domain of p73 binds to the N terminus of MDM2

• Published in: Biochimica et biophysica acta. General subjects Vol. 1863; no. 4; pp. 760 - 770
• Main Authors: Neira, José L., Díaz-García, Clara, Prieto, Manuel, Coutinho, Ana
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-04-2019
• Subjects: Binding; Fluorescence; MDM2; NMR; SAM; Binding; CD; CSP; UV; SDS; Fluorescence; α4α5; PAGE; TA; SEC; TSP; NMR; p73α; SAMp73; MDM2; IPTG; IRF; TCEP; SAM; BLI
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2019.01.019

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.
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).
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.
A new interaction between MDM2 and SAMp73 has been found, which could have potential therapeutic applications in cancers involving inactivated p53.
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.
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•SAMp73 bound to N terminus of MDM2.•Binding was ~ 10 μM., as shown by fluorescence, NMR and interferometry.•Binding involved mainly the last helix of SAMp73.