Al Kafri Nour, Ahnström Josefin, Teraz-Orosz Adrienn, Chaput Ludovic, Singh Natesh, Villoutreix Bruno O, Hafizi Sassan
School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth, UK.
Faculty of Medicine, Dept. of Immunology and Inflammation, Imperial College London, UK.
Biochem Biophys Rep. 2022 Apr 28;30:101263. doi: 10.1016/j.bbrep.2022.101263. eCollection 2022 Jul.
The homologous proteins Gas6 and protein S (ProS1) are both natural ligands for the TAM (Tyro3, Axl, MerTK) receptor tyrosine kinases. ProS1 selectively activates Tyro3; however, the precise molecular interface of the ProS1-Tyro3 contact has not been characterised. We used a set of chimeric proteins in which each of the -terminal laminin G-like (LG) domains of ProS1 were swapped with those of Gas6, as well as a set of ProS1 mutants with novel added glycosylations within LG1. Alongside wildtype ProS1, only the chimera containing ProS1 LG1 domain stimulated Tyro3 and Erk phosphorylation in human cancer cells, as determined by Western blot. In contrast, Gas6 and chimeras containing minimally the Gas6 LG1 domain stimulated Axl and Akt phosphorylation. We performed homology modelling and molecular docking analysis to construct and evaluate structural models of both ProS1-Tyro3 and Gas6-Axl ligand-receptor interactions. These analyses revealed a contact between the ProS1 LG1 domain and the first immunoglobulin domain of Tyro3, which was similar to the Gas6-Axl interaction, and involved long-range electrostatic interactions that were further stabilised by hydrophobic and polar contacts. The mutant ProS1 proteins, which had added glycosylations within LG1 but which were all outside of the modelled contact region, all activated Tyro3 in cells with no hindrance. In conclusion, we show that the LG1 domain of ProS1 is necessary for activation of the Tyro3 receptor, involving protein-protein interaction interfaces that are homologous to those of the Gas6-Axl interaction.
同源蛋白Gas6和蛋白S（ProS1）都是TAM（Tyro3、Axl、MerTK）受体酪氨酸激酶的天然配体。ProS1选择性激活Tyro3；然而，ProS1与Tyro3接触的精确分子界面尚未得到表征。我们使用了一组嵌合蛋白，其中ProS1的每个N端层粘连蛋白G样（LG）结构域都与Gas6的相应结构域进行了交换，以及一组在LG1内添加了新糖基化的ProS1突变体。与野生型ProS1一起，通过蛋白质印迹法测定，只有包含ProS1 LG1结构域的嵌合体在人癌细胞中刺激了Tyro3和Erk磷酸化。相比之下，Gas6和至少包含Gas6 LG1结构域的嵌合体刺激了Axl和Akt磷酸化。我们进行了同源建模和分子对接分析，以构建和评估ProS1-Tyro3和Gas6-Axl配体-受体相互作用的结构模型。这些分析揭示了ProS1 LG1结构域与Tyro3的第一个免疫球蛋白结构域之间的接触，这与Gas6-Axl相互作用相似，并且涉及通过疏水和极性接触进一步稳定的长程静电相互作用。在LG1内添加了糖基化但都在模拟接触区域之外的突变ProS1蛋白，在细胞中均无障碍地激活了Tyro3。总之，我们表明ProS1的LG1结构域是激活Tyro3受体所必需的，涉及与Gas6-Axl相互作用同源的蛋白质-蛋白质相互作用界面。
