p73 的 C 端 SAM 结构域与 MDM2 的 N 端结合。

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

机构信息

Instituto 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.

CQFM-IN and iBB- Institute for Bioengineering and Bioscience, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal.

出版信息

Biochim Biophys Acta Gen Subj. 2019 Apr;1863(4):760-770. doi: 10.1016/j.bbagen.2019.01.019. Epub 2019 Feb 5.

DOI:10.1016/j.bbagen.2019.01.019

PMID:30735716

Abstract

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, T-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.

摘要

背景

p53、p63 和 p73 蛋白属于 p53 转录因子家族，在肿瘤抑制中发挥关键作用。p73 的 α-剪接变体（p73α）在其 C 端具有一个无活性的 α 基序（SAM）；这个由五个螺旋（α1 到 α5）组成的结构域，SAMp73，被认为介导蛋白质-蛋白质相互作用。E3 连接酶 MDM2 与 p73 的 N 端反式激活结构域（TA）结合，但它不通过泛素化促进其降解；然而，这种 MDM2/p73 相互作用的细节尚不完全清楚。

方法

我们使用几种生物物理技术，即荧光、远紫外圆二色性（CD）、NMR 和生物层干涉（BLI），研究了 SAMp73 与 N 端 MDM2 的结合。

结果

我们通过荧光、T-弛豫测量和 BLI 获得的结果表明，两种蛋白质之间存在结合，解离常数约为 10 μM。此外，HSQC-NMR 显示，SAMp73 的结合区域主要涉及主要α-螺旋α5 及其附近的α4 残基。这种结合非常特异，包含 SAMp73 的α4 和α5 螺旋的分离肽，α4α5，也与 MDM2 的 N 端结合，尽管亲和力比整个结构域弱。