Melnikova Daria, Bogdanov Ivan, Ovchinnikova Tatiana, Finkina Ekaterina
Science-Educational Center, M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia.
Department of Physicochemical Biology and Biotechnology, Moscow Institute of Physics and Technology (State University), 141701 Dolgoprudny, Russia.
Membranes (Basel). 2020 Nov 20;10(11):357. doi: 10.3390/membranes10110357.
It is known that plant lipid transfer proteins (LTPs) bind a broad spectrum of ligands including fatty acids (FAs), phospho- and glycolipids, acyl-coenzyme A and secondary metabolites. In this work, we used protein-lipid overlay assays to identify new putative LTP ligands. In our experiments, the lentil lipid transfer protein Lc-LTP2 as well as LTPs from other plants were shown to bind phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2). Molecular modeling, 2-p-toluidinonaphthalene-6-sulphonate (TNS) displacement and liposome leakage experiments with Lc-LTP2 and its mutant analogs (R45A, Y80A, R45A/Y80A) were performed to investigate interactions between the protein and PI(4,5)P2. It was shown that PI(4,5)P2 initially interacted with the "bottom" entrance of the protein cavity and after complex formation the large polar head of this ligand was also oriented towards the same entrance. We also found that two highly conserved residues in plant LTPs, Arg45 and Tyr80, played an important role in protein-ligand interactions. Apparently, Arg45 is a key residue for interaction with PI(4,5)P2 during both initial contacting and holding in the protein cavity, while Tyr80 is probably a key amino acid playing an essential role in Lc-LTP2 docking to the membrane. Thus, we assumed that the ability of Lc-LTP2 to bind PI(4,5)P2 suggests the involvement of this protein in plant signal transduction.
已知植物脂质转移蛋白（LTPs）能结合多种配体，包括脂肪酸（FAs）、磷酸和糖脂、酰基辅酶A以及次生代谢产物。在本研究中，我们利用蛋白质 - 脂质覆盖分析来鉴定新的潜在LTP配体。在我们的实验中，扁豆脂质转移蛋白Lc - LTP2以及来自其他植物的LTPs被证明能结合磷脂酰肌醇（4,5）-二磷酸（PI(4,5)P2）。进行了分子建模、2 - 对甲苯胺基萘 - 6 - 磺酸盐（TNS）置换以及用Lc - LTP2及其突变类似物（R45A、Y80A、R45A/Y80A）进行的脂质体泄漏实验，以研究该蛋白与PI(4,5)P2之间的相互作用。结果表明，PI(4,5)P2最初与蛋白腔的“底部”入口相互作用，复合物形成后，该配体的大极性头部也朝向同一入口。我们还发现植物LTPs中的两个高度保守残基Arg45和Tyr80在蛋白 - 配体相互作用中起重要作用。显然，Arg45是在初始接触和在蛋白腔内结合过程中与PI(4,5)P2相互作用的关键残基，而Tyr80可能是在Lc - LTP2与膜对接中起关键作用的氨基酸。因此，我们推测Lc - LTP2结合PI(4,5)P2的能力表明该蛋白参与植物信号转导。
