Fig. 4: L704 is part of the extracellular gate of ABCG36 and L704F increases free energy surface for camalexin in the entrance region.
a Chemical formulas for potential ABCG36 substrates employed in this study (downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov)). b THs of ABCG36 are shown from the extracellular space. Extracellular gate residues are highlighted by sticks and balls. Docked IBA is shown using stick representation. Electronic structural deformation, the electric field lines of IBA, CLX, and IAA before (c) and after binding to the ABCG36 substrate pocket (d). The electronic binding energies and the results displayed here were obtained by ab initio calculations using DFT methods. e, f Specific 3H-IBA and 3H-CLX binding to indicated Arabidopsis microsomes calculated as the difference between total and unspecific substrate binding determined in the absence (total) and presence of a 1000-fold excess of non-radiolabeled substrate concentrations (unspecific), respectively. Significant differences (p < 0.05) of means ± SE (n = 3 independent microsomal preparations with each 3 technical replica) to the corresponding Wt were determined using Ordinary One-way ANOVA (Tukey’s multiple comparison test) and are indicated by different lowercase letters. Data are presented as box-and-whisker plots, where median and 25th and 75th percentiles are represented by the box itself and the middle line, respectively; means are indicated by a “+”. Source data are provided as a Source Data file. Free energy surfaces (FES) were calculated from metadynamics simulations with complexes of IBA (g) and CLX (h) with Wt and L704F ABCG36, respectively. Pulling of IBA (i) and CLX (j) from the central binding pocket to the extracellular space were performed in biased simulations with Wt and L704F ABCG36, respectively. Frequencies of contacts between the small molecules and the protein are plotted.
