Polo Andrea, Colonna Giovanni, Guariniello Stefano, Ciliberto Gennaro, Costantini Susan
Servizio di Informatica Medica, Azienda Ospedaliera Universitaria, Seconda Università di Napoli, Napoli, Italy.
Mol Biosyst. 2016 Mar;12(3):758-72. doi: 10.1039/c5mb00679a. Epub 2016 Jan 6.
The intrinsically disordered proteins (IDPs) cannot be described by a single structural representation but, due to their high structural fluctuation, through conformational ensembles. Certainly, molecular dynamics (MD) simulations represent a useful tool to study their different conformations capturing the conformational distribution. Our group is focusing on the structural characterization of proteins belonging to the seleno-proteome due to their involvement in cancer. They present disordered domains central for their biological function, and, in particular, SELK is a single-pass transmembrane protein that resides in the endoplasmic reticulum membrane (ER) with a C-terminal domain exposed to the cytoplasm that is known to interact with different components of the endoplasmic reticulum associated to the protein degradation (ERAD) pathway. This protein is found to be up-expressed in hepatocellular carcinoma and in other cancers. In this work we performed a detailed analysis of the C-terminal domain sequence of SELK and discovered that it is characterized by many prolines, and four negatively and eleven positively charged residues, which are crucial for its biological activity. This region can be considered as a weak polyelectrolyte and, specifically, a polycation, with high disordered propensity and different phosphorylation sites dislocated along the sequence. Then, we modeled its three-dimensional structure by performing MD simulations in water at neutral pH to analyze the structural stability as well as to identify the presence of HUB residues that play a key structural role as evidenced by the residue-residue interaction network analysis. Through this approach, we demonstrate that the C-terminal domain of SELK (i) presents a poor content of regular secondary structure elements, (ii) is dynamically stabilized by a network of intra-molecular H-bonds and H-bonds with water molecules, (iii) is highly fluctuating and, therefore, can be described only through a conformational ensemble, where we evidenced a distribution of equilibrium conformers which continuously inter-change their conformations. Finally to verify the specific role played by the negative charges, we also performed MD simulations at acidic pH. Overall, all the obtained results evidenced that SELK has the dynamic structural features to be defined as a HUB protein able to interact with multiple members. Therefore, considering the possible role that this protein can have in cancer development and progression, it can represent a target for drug design studies.
内在无序蛋白（IDPs）无法用单一的结构表示来描述，而是由于其高度的结构波动，通过构象集合来描述。当然，分子动力学（MD）模拟是研究其不同构象、捕捉构象分布的有用工具。我们的研究小组专注于硒蛋白组中蛋白质的结构表征，因为它们与癌症有关。它们具有对其生物学功能至关重要的无序结构域，特别是，SELK是一种单次跨膜蛋白，位于内质网膜（ER）中，其C端结构域暴露于细胞质中，已知该结构域与内质网相关蛋白降解（ERAD）途径的不同成分相互作用。这种蛋白在肝细胞癌和其他癌症中被发现高表达。在这项工作中，我们对SELK的C端结构域序列进行了详细分析，发现它的特征是有许多脯氨酸，以及四个带负电荷和十一个带正电荷的残基，这些对其生物学活性至关重要。该区域可被视为一种弱聚电解质，具体而言，是一种聚阳离子，具有高无序倾向和沿序列分布的不同磷酸化位点。然后，我们通过在中性pH的水中进行MD模拟来构建其三维结构模型，以分析结构稳定性，并识别通过残基-残基相互作用网络分析证明起关键结构作用的枢纽残基的存在。通过这种方法，我们证明SELK的C端结构域：（i）规则二级结构元件的含量较低；（ii）通过分子内氢键和与水分子的氢键网络动态稳定；（iii）高度波动，因此只能通过构象集合来描述，我们证明了平衡构象的分布，这些构象不断相互交换构象。最后，为了验证负电荷所起的特定作用，我们还在酸性pH下进行了MD模拟。总体而言，所有获得的结果都证明SELK具有被定义为能够与多个成员相互作用的枢纽蛋白的动态结构特征。因此，考虑到这种蛋白在癌症发生和发展中可能具有的作用，它可以成为药物设计研究的一个靶点。
