IDIBE, Universidad Miguel Hernández, 03202 Elche, Alicante, Spain; Instituto de Biocomputación y Física de Sistemas Complejos (BIFI), Joint Units IQFR-CSIC-BIFI, GBsC-CSIC-BIFI, Universidad de Zaragoza, 50009 Zaragoza, Spain.
Instituto de Biocomputación y Física de Sistemas Complejos (BIFI), Joint Units IQFR-CSIC-BIFI, GBsC-CSIC-BIFI, Universidad de Zaragoza, 50009 Zaragoza, Spain; CNR-NANOTEC, Licryl-UOS Cosenza and CEMIF.Cal, Department of Physics, University of Calabria, 87036 Rende, Italy.
Biochim Biophys Acta Gen Subj. 2021 Jul;1865(7):129914. doi: 10.1016/j.bbagen.2021.129914. Epub 2021 Apr 17.
Plakophilin 1 (PKP1) is a component of desmosomes, which are key structural components for cell-cell adhesion, and can also be found in other cell locations. The p53, p63 and p73 proteins belong to the p53 family of transcription factors, playing crucial roles in tumour suppression. The α-splice variant of p73 (p73α) has at its C terminus a sterile alpha motif (SAM); such domain, SAMp73, is involved in the interaction with other macromolecules.
We studied the binding of SAMp73 with the armadillo domain of PKP1 (ARM-PKP1) in the absence and the presence of 100 mM NaCl, by using several biophysical techniques, namely fluorescence, far-ultraviolet circular dichroism (CD), nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC), and molecular docking and simulations.
Association was observed between the two proteins, with a dissociation constant of ~5 μM measured by ITC and fluorescence in the absence of NaCl. The binding region of SAMp73 involved residues of the so-called "middle-loop-end-helix" binding region (i.e., comprising the third helix, together with the C terminus of the second one, and the N-cap of the fourth), as shown by N, H- HSQC-NMR spectra. Molecular modelling provided additional information on the possible structure of the binding complex.
This newly-observed interaction could have potential therapeutic relevance in the tumour pathways where PKP1 is involved, and under conditions when there is a possible inactivation of p53.
The discovery of the binding between SAMp73 and ARM-PKP1 suggests a functional role for their interaction, including the possibility that SAMp73 could assist PKP1 in signalling pathways.
桥粒磷蛋白 1（PKP1）是桥粒的组成部分，桥粒是细胞间黏附的关键结构成分，也存在于其他细胞位置。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 末端具有一个无活性的α基序（SAM）；这种结构域，SAMp73，参与与其他大分子的相互作用。
我们使用几种生物物理技术，即荧光、远紫外圆二色性（CD）、核磁共振（NMR）、等温滴定量热法（ITC）和分子对接和模拟，研究了 SAMp73 与 PKP1 的卷曲螺旋结构域（ARM-PKP1）在无和存在 100mM NaCl 下的结合情况。
在无 NaCl 时，通过 ITC 和荧光测量，观察到两种蛋白质之间存在关联，解离常数约为 5μM。SAMp73 的结合区域涉及所谓的“中环端螺旋”结合区域的残基（即包含第三螺旋，以及第二螺旋的 C 末端和第四螺旋的 N 端帽），如 N、H-HSQC-NMR 谱所示。分子建模提供了关于结合复合物可能结构的附加信息。
这种新观察到的相互作用可能在涉及 PKP1 的肿瘤途径中具有潜在的治疗意义，并且在可能失活 p53 的情况下。
SAMp73 和 ARM-PKP1 之间结合的发现表明它们相互作用具有功能作用，包括 SAMp73 可能协助 PKP1 信号通路的可能性。
