Fig. 2

The in silico and Biacore analysis of the molecular interactions within the GHCer/TRAX complex. A The TRAX protein had seven alpha helices (α1–α7). The carbon chains of sphingosine (SPH; orange) and fatty acid (FA; black) of GHCer bound to a hydrophobic groove in TRAX (green surface). On the other hand, the glycan of GHCer interacted mainly with a hydrophilic region in TRAX (yellow surface). The amino acid residues in these amphipathic binding sites were labeled. B MD simulation was performed for assembling the GHCer/TRAX complex. Various GHCer conformers associated with the binding sites on TRAX by changing its conformation during MD simulation and finally formed a stable complex at equilibrium. The right panel shows the model of stable complex with 90° rotation. C The model of GHCer/TRAX complex at equilibrium (20 ns) showed that potentially as many as seven H-bonds could take place between GHCer and TRAX as predicted by MD simulation. When the conformers finally reached equilibrium, the molecular trajectories of MD simulation indicated that the number of H-bonds fluctuated and the average number of H-bonds between GHCer and TRAX was 4.6 ± 1.3. Furthermore, the SSEA3Cer/TRAX complex was generated from GHCer/TRAX model by replacing the Fuc molecule with a hydrogen atom. During MD simulation, the number of H-bonds between SSEA3Cer and TRAX decreased with time due to the conformational changes of SSEA3Cer. D MD simulation displayed poor interaction between GHCer and mutant form of TRAX (Mut TRAX with Q223A and Q219A). Only sugar #1 of GHCer formed H-bond with E216 of TRAX. E The CM5 chips were coated with mAb VK9 for Biacore assays. GHCer was then captured by the chip, followed by analyzing binding responses of various concentrations of wild-type TRAX or Mut TRAX