title: Understanding the roles of Lys33 and Arg45 in the binding-site stability of LjLTP10, an LTP related to drought stress in Lotus japonicus.
abstract: In Lotus japonicus, as in most plants, long-chain fatty acids are important components of cuticular wax, one of the principal functions of which is to act as a barrier to water loss in response to drought stress. It is thought that lipid transfer proteins (LTPs) are involved in the process of cuticle formation. We previously described LjLTP10 as an LTP involved in cuticle formation during acclimation response to drought stress in L. japonicus. The structural model of LjLTP10 had two residues (K33 and R45) in the hydrophobic cavity, although the role of these residues was unclear. In the present work, we investigated the molecular mechanism involved in the transport of lipid precursors in L. japonicus and clarified the importance of the residues K33 and R45. First, in silico site-directed mutagenesis studies were carried out on the LjLTP10 structure. Structural analysis showed that LjLTP10 mutants possess similar structures but their hydrophobic cavities are somewhat different. Unfavorable energies for the interactions of the mutant proteins with different ligands were found by molecular docking and molecular dynamics simulations. We also examined the contributions of energetic parameters to the free energy of the protein-ligand complex using the MM-GBSA method. Results showed that the different complexes present similar, favorable van der Waals interactions, whereas electrostatic interactions were not favored in the mutant structures. Our study indicates that the residues K33 and R45 play a crucial role in maintaining the binding pocket structure required for lipid transport.
authors.affiliation: Escuela de Ingeniería en Bioinformática, Facultad de Ingeniería, Universidad de Talca, Casilla 747, Talca, Chile.
authors.forename: Felipe
authors.initials: F
authors.lastname: Valenzuela-Riffo
authors.affiliation: Unidad de Recursos Genéticos, Instituto de Investigaciones Agropecuarias, INIA-Quilamapu, Chillán, Chile. gtapia@inia.cl.
authors.forename: Gerardo
authors.initials: G
authors.lastname: Tapia
authors.affiliation: Laboratorio de Fisiología Vegetal y Genética Molecular, Instituto de Ciencias Biológicas, Universidad de Talca, Casilla 747, Talca, Chile.
authors.forename: Carolina
authors.initials: C
authors.lastname: Parra-Palma
authors.affiliation: Laboratorio de Fisiología Vegetal y Genética Molecular, Instituto de Ciencias Biológicas, Universidad de Talca, Casilla 747, Talca, Chile. lmorales@utalca.cl.
authors.forename: Luis
authors.initials: L
authors.lastname: Morales-Quintana
mesh_terms: D001120:Arginine / Q000737:chemistry*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D055864:Droughts; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D000070116:Lotus / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D008239:Lysine / Q000737:chemistry*; D008958:Models, Molecular; D008968:Molecular Conformation; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D055550:Protein Stability; D021281:Quantitative Structure-Activity Relationship; D013312:Stress, Physiological
chemical_list: D002352:Carrier Proteins; D008024:Ligands; D010940:Plant Proteins; C030363:lipid transfer protein; D001120:Arginine; D008239:Lysine
keywords: In silico site-directed mutagenesis; Lipid transfer protein; Lotus japonicus; MM-GBSA; Molecular dynamics simulations