Lipid transfer proteins (LTPs) are an important class of plant proteins containing an internal cavity and binding hydrophobic ligands. Although LTP structures and functions are well studied, mechanisms of ligand binding remain unclear. Earlier, we discovered the lentil lipid transfer protein Lc-LTP2 capable of binding and transfer various ligands. We have shown that the “bottom” entrance of the Lc-LTP2 cavity takes part in attachment to the micelle surface and in lipids uptake. Here, we studied the role of Arg45 and Tyr80, located at the “bottom” entrance, in Lc-LTP2 ligand binding. We obtained recombinant mutant analogs of Lc-LTP2 (R45A, Y80A, R45A/Y80A), investigated their ability to bind fatty acids and lysolipids, as well as performed molecular modeling of the protein-ligand complexes. We showed that replacement of one or both residues led to a change of the internal hydrophobic cavity dimensions. As a result, lipids may change their orientation into the protein cavity, and thereby binding ability of mutant analogs may be affected as well. In the present work, we revealed an important role of Arg45 and Tyr80 in stabilization of the Lc-LTP2 complexes with both fatty acids and lysolipids with different ligand orientation.

脂质转移蛋白（LTP）是一类重要的植物蛋白，包含内部空腔并结合疏水性配体。尽管对LTP的结构和功能进行了很好的研究，但配体结合的机制仍不清楚。早些时候，我们发现了能够结合和转移各种配体的扁豆脂质转移蛋白Lc-LTP2。我们已经表明，Lc-LTP2腔的“底部”入口参与了对胶束表面的附着和脂质的吸收。在这里，我们研究了位于“底部”入口的Arg45和Tyr80在Lc-LTP2配体结合中的作用。我们获得了Lc-LTP2的重组突变体类似物（R45A，Y80A，R45A / Y80A），研究了它们与脂肪酸和溶血脂结合的能力，并对蛋白质-配体复合物进行了分子建模。我们表明替换一个或两个残基导致内部疏水腔尺寸的变化。结果，脂质可能改变其进入蛋白质腔的方向，从而也可能影响突变类似物的结合能力。在目前的工作中，我们揭示了Arg45和Tyr80在稳定Lc-LTP2与脂肪酸和溶血脂具有不同的配体取向的复合物中的重要作用。