Abstract
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.
Highlights
Lipids play an especially important role in plant cells
We showed that PI(4,5)P2, mainly located in plasmatic membranes and regulating cellular functions through multiple mechanisms, is a novel signal ligand of the lipid transfer protein Lc-LTP2 from lentil seeds
We showed that PI(4,5)P2 is a new possible ligand of lentil Lc-LTP2 and other plant lipid transfer proteins (LTPs) using protein-lipid overlay assays
Summary
Lipids play an especially important role in plant cells. They function as barriers, and take part in signal transduction, trafficking, morphological changes, and cell division via lipid-protein interactions. Many lipid-binding proteins have been identified in plants, including lipid transfer proteins (LTPs). LTPs are small, cationic proteins that are found in all land plants, encoded by large gene families, and expressed in most tissues [1,2]. Their abundance indicates a particular importance in plant life. In LTPs, four conserved disulfide bridges stabilize compact spatial structures containing three or four α-helices and a central hydrophobic cavity suitable for binding varied hydrophobic ligands [3]
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